Reconstituteable dried powder formulation and method of use thereof

Abstract

This disclosure is intended for the use of reconstituted mRNA dry powder particles for parenteral administration. This disclosure is also intended for a method of producing dry powder particles supplemented with appropriate excipients for optimal thermal stability and in vivo expression.

Description

[Technical Field]

[0001] Messenger RNA therapy (MRT) is an important approach for treating a variety of diseases. The use of in vitro transcribed mRNA for protein production in animals was first reported in the 1990s. With advances in biotechnology, the stability and immunogenicity of mRNA have been optimized for usefulness in human clinical settings. However, safe and efficient in vivo delivery remains a major obstacle in the development of mRNA therapeutics.

[0002] Dried powder formulations of mRNA (including inhalation delivery to the lungs) can be useful and advantageous. Reconstituted dried powder formulations can offer additional advantages, such as improved formulations useful for other administration routes, such as parenteral administration. In particular, overcoming the challenge of developing both the thermal stability and reconstituted dried powder formulations of mRNA-LNPs has presented challenges, for example, in the development of dried powder formulations suitable for parenteral administration. Improved dried powder formulations, including reconstituted ones, are still needed. [Overview of the Initiative] [Means for solving the problem]

[0003] This specification describes dry powder formulations that can offer specific advantages and improvements. For example, the dry powder formulations provided herein are reconstituteable and therefore suitable for a variety of uses and can offer a variety of therapeutic benefits. Methods for preparing and reconstituting dry powder formulations, as well as methods for using dry powder formulations, are also described herein.

[0004] In some embodiments, the Disclosure comprises a dry powder formulation for reconstitution, comprising (a) an excipient which is sucrose or trehalose; and (b) lipid nanoparticles (LNPs) containing messenger RNA (mRNA) encapsulated by one or more lipids, wherein the weight (w / w) ratio of the excipient in (a) to the total lipids in the LNPs in (b) is at least about 5. In some embodiments, the excipient is sucrose. In some embodiments, the excipient is trehalose. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNPs is at least about 5.6, 11, or 15. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNPs is at least about 11.1 or at least about 15.6. In some embodiments, the LNPs comprise one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

[0005] In some embodiments, the Disclosure encompasses a dry powder formulation for reconstitution comprising (a) an excipient which is a sugar or sugar alcohol; and (b) lipid nanoparticles (LNPs) containing messenger RNA (mRNA) encapsulated by one or more lipids, wherein the w / w ratio of the excipient in (a) to the total lipids in the LNPs in (b) is at least about 5. In some embodiments, the sugar or sugar alcohol is selected from sucrose, mannitol, xylitol, lactose, and trehalose. In some embodiments, the sugar or sugar alcohol is trehalose. In some embodiments, the sugar or sugar alcohol is sucrose. In some embodiments, the w / w ratio of the sugar or sugar alcohol to the total lipids in the LNPs is at least about 5.6, 11, or 15. In some embodiments, the w / w ratio of the sugar or sugar alcohol to the total lipids in the LNPs is at least about 11.1 or at least about 15.6. In some embodiments, the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

[0006] In some embodiments, the particle size (e.g., average particle size) of the reconstituted dry powder formulation is less than approximately 120 nm. In some embodiments, the encapsulation rate is greater than 60%. In some embodiments, the particle size (e.g., average particle size) of the dry powder is less than 5 μm. In some embodiments, the dry powder formulation is stable after long-term storage, for example, at 2-8°C (e.g., 4°C). In some embodiments, the mRNA maintains 80% or more integrity after storage, for example, at 2-8°C (e.g., 4°C) for at least 6 months. In some embodiments, the mRNA maintains 80% or more integrity after storage for up to 1 year at 2-8°C (e.g., 4°C). In some embodiments, the mRNA maintains 80% or more integrity after storage for at least 1 year at 2-8°C (e.g., 4°C). In some embodiments, the mRNA maintains approximately 80% or more integrity after storage for more than 1 year at 2-8°C (e.g., 4°C).

[0007] In some embodiments, the mRNA encodes a therapeutic protein. In some embodiments, the mRNA encodes an antigen. In some embodiments, the formulation is suitable as a vaccine. In some embodiments, the formulation, when reconstituted, is suitable for parenteral delivery. In some embodiments, the formulation, when reconstituted, is suitable for intramuscular, intravenous, or subcutaneous delivery. In some embodiments, the formulation, when reconstituted, is suitable for mucosal delivery. In some embodiments, the formulation, when reconstituted, is suitable for oral, sublingual, or intranasal delivery.

[0008] In some embodiments, the Disclosure includes a method for delivering mRNA in vivo, comprising administering a reconstituted form of the dried powder formulation of the Disclosure to a subject in need thereof. In some embodiments, the Disclosure includes a method for treating a disease or disorder in a subject, comprising administering a reconstituted form of the dried powder formulation of the Disclosure to the subject. In some embodiments, the reconstituted form of the dried powder formulation is administered subcutaneously, intravenously, or intramuscularly.

[0009] In some embodiments, the Disclosure includes a reconstituted form of the dried powder formulation of the Disclosure for use in the treatment of a disease or disorder in a subject. In some embodiments, the Disclosure includes the use of the reconstituted form of the dried powder formulation of the Disclosure in the manufacture of a pharmaceutical product for the treatment of a disease or disorder in a subject. In some embodiments, the reconstituted form of the dried powder formulation is formulated for subcutaneous, intravenous, or intramuscular administration.

[0010] In some embodiments, the present disclosure comprises a method for preparing a reconstituted dry powder formulation, comprising: (a) combining a first mixture comprising lipid nanoparticles (LNPs) with an ethanol solution to obtain a second mixture, wherein the LNPs comprise messenger RNA (mRNA) encapsulated by one or more lipids, and the ethanol solution comprises an excipient, sucrose or trehalose, at a concentration of about 1-11% (w / v); and (b) spray-drying the second mixture to obtain a dry powder formulation.

[0011] In some embodiments, the Disclosure encompasses a method for preparing a reconstituted dry powder formulation, comprising (a) providing a dry powder formulation prepared according to the Disclosure, and (b) reconstituting the dry powder formulation in water or a buffer to obtain a reconstituted dry powder formulation. In some embodiments, the LNPs in the reconstituted dry powder formulation have a diameter of about 100 nm. In some embodiments, the weight ratio (w / w) of sucrose or trehalose to total lipids in the LNPs is at least about 5, at least about 11, or at least about 15. In some embodiments, the concentration of sucrose or trehalose after reconstitution is greater than 2% (w / v), greater than 5% (w / v), or greater than 7% (w / v). In some embodiments, the concentration of sucrose or trehalose after reconstitution is about 2% (w / v) to about 10% (w / v). In some embodiments, the particle size of the reconstituted dry powder formulation (e.g., the average particle size of the LNPs in the reconstituted formulation) is less than 120 nm. In some embodiments, the reconstituted LNP particle size (e.g., the average particle size of LNPs in the reconstituted formulation) is 80–120 nm. In some embodiments, the reconstituted LNP particle size (e.g., the average particle size of LNPs in the reconstituted formulation) is 80–115 nm. [Brief explanation of the drawing]

[0012] [Figure 1A-1B] The images show the dried powder products (DPP) produced using mRNA-LNP formulations containing xylitol and lactose, respectively, when the excipients were not sufficiently spray-dried. The liquid formulations adhered to the cyclone separator, and the dried powder products were not collected in the collection container. [Figure 2A] As described in Example 2, aggregates are observed in the reconstituted dry powder (DP) using 7% (w / v) mannitol (right) as an excipient at a magnification of 400 times, compared to water alone (left). [Figure 2B] The appearance of the reconstituted dried powders (right) using 7% (left), 5% (center), and 2.5% (w / v) mannitol as excipients, as described in Example 2, is shown. [Figure 3A] As described in Example 2, no aggregates were observed in the reconstituted DP using 5% (w / v) sucrose as an excipient (right) under a microscope, compared to water alone (left). [Figure 3B] The appearance of the reconstituted dry powders (right) using 7% (left), 5% (center), and 2.5% (w / v) sucrose as excipients, as described in Example 2, is shown. [Figure 3C] As described in Example 2, the DPP produced using an mRNA-LNP formulation with 2.5% (w / v) sucrose as an excipient was not sufficiently spray-dried, and the liquid formulation adhered to the cyclone separator. [Figure 4A] The appearance of the reconstituted dried powders using 7% (right), 5% (center), and 2.5% (w / v) trehalose (left) as excipients, as described in Example 2, is shown. [Figure 4B] The appearance of the reconstituted dried powder using 1.25% (w / v) trehalose as an excipient is shown, as described in Example 2. [Figure 4C] As described in Example 2, compared to water alone (left), no aggregates were observed in the reconstituted DP using 2.5% (w / v) trehalose as an excipient (right) under a microscope. [Figure 4D] As described in Example 2, compared to the mRNA-LNP preparation using 7% (w / v) trehalose as an excipient (right), the DPP produced using the mRNA-LNP preparation using 1.25% (w / v) trehalose as an excipient did not spray dry sufficiently, and most of the liquid preparation adhered to the cyclone separator, with only a small amount of dried powder being recovered in the collection container (center). [Figure 5] The appearance of the reconstituted dry powder using trehalose added as part of the reconstitution solvent, as described in Example 2, is shown. The concentrations shown (7% and 5% (w / v)) are the concentrations of trehalose in the reconstituted composition. [Figure 6A-6B]These graphs show the changes in particle size (Figure 6A), polydispersity index (PDI; Figure 6B), encapsulation efficiency (EE; Figure 6C), and mRNA integrity (Figure 6D) of the reconstituted composition using 5% (w / v) trehalose over a 12-month period at various temperatures (25°C, 4°C, or -20°C). [Figure 6C-6D] Same as above. [Figure 7A] This shows the mRNA integrity of the reconstituted composition using 5% (w / v) trehalose, measured by capillary electrophoresis at 25°C, 4°C, and -20°C, respectively, after 9 months of storage. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] This shows the mRNA integrity of the reconstituted composition using 5% (w / v) trehalose, measured by capillary electrophoresis at 25°C, 4°C, and -20°C, respectively, after 6 months of storage. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] This shows the mRNA integrity of the reconstituted composition using 5% (w / v) trehalose, measured by capillary electrophoresis at 25°C, 4°C, and -20°C, respectively, after 3 months of storage. [Figure 7H] Same as above. [Figure 7I] Same as above. [Figure 7J] Capillary electrophoresis plots of mRNA standards used as controls are shown. [Figure 8A] As described in Example 5, this graph shows that trehalose dried powder ("trehalose DP") does not show any change in mRNA integrity after spray drying compared to control mRNA ("standard") after reconstitution. [Figure 8B] This graph shows OTC expression in mice after intravenous injection of liquid control and reconstituted trehalose dry powder. [Figure 9]This graph shows human erythropoietin (hEPO) expression in mice after intramuscular injection of liquid control and reconstituted trehalose dry powder. [Figure 10A-10B] Figure 10A shows graphs indicating no significant changes in particle size, PDI, inclusion efficiency, and mRNA integrity for three different DPPs after storage at 2–8°C for 12 months, as described in Example 8. Figure 10A also shows no significant changes in hEPO expression over 12 months. Figure 10A: DPP containing mRNA encoding hEPO ("cKK-E10-hEPO dry powder"); Figure 10B: DPP containing mRNA encoding influenza antigen ("cKK-E10-moono-Flu dry powder"); Figure 10C: DPP containing mRNA encoding antigen derived from respiratory syncytial virus (RSV) antigen ("cKK-E10-RSV dry powder"). [Figure 10C] Same as above. [Figure 11A] This graph shows that there were no significant changes in particle size, PDI, inclusion efficiency, and mRNA integrity for DPP prepared using four different influenza mRNAs (QIV-Flu) after being stored at 2-8°C for 10 months, as described in Example 8. [Figure 11B] Capillary electrophoresis profiles of QIV-Flu mRNA ("QIV mRNA"; top), mRNA extracted from reconstituted DPP in T0 ("Dried Powder T0"; center), and mRNA extracted from reconstituted DPP after storage at 2–8°C for 5.5 months ("Dried Powder 5.5 Months"; bottom) are shown. [Figure 11B-1] Same as above. [Figure 11B-2] Same as above. [Modes for carrying out the invention]

[0013] definition To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of the following terms and other terms are provided throughout this specification. Publications and other reference materials referenced herein to provide background to this disclosure and to offer additional details relating to its implementation are incorporated herein by reference.

[0014] Approximately or about: As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​that are similar to the stated reference values. In certain embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “about” refer to values ​​in the range of (including all values ​​and subranges between them) that fall within 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the stated reference values ​​(greater than or less than those values), except where such numbers exceed 100% of the possible values. In exemplary embodiments, approximately 5 refers to 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, and 5.5.

[0015] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, mRNA delivery encompasses situations in which mRNA is delivered to a target tissue, the encoded protein is expressed, and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which mRNA is delivered to a target tissue, the encoded protein is expressed, secreted into the patient’s circulatory system (e.g., serum), systematically distributed, and absorbed by other tissues (also referred to as “systemic distribution” or “systemic delivery”). In some embodiments, delivery is oral, intramuscular, intravenous, subcutaneous, sublingual, or oral delivery.

[0016] Encapsulation: As used herein, the term “encapsulation,” or its grammatical equivalent, refers to the process of confining nucleic acid molecules within nanoparticles.

[0017] Encapsulation Efficiency: As used herein, “Encapsulation Efficiency” or “EE” refers to the amount of therapeutic and / or prophylactic agent, such as the ribodiffusing molecules of this disclosure, that becomes part of the lipid nanoparticles (LNPs), compared to the initial total amount of therapeutic and / or prophylactic agent used in the preparation of the LNPs. For example, if 97 mg of therapeutic and / or prophylactic agent is encapsulated in the LNPs out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency may be given as 97%. Encapsulation efficiency may be determined, for example, by a RiboGreen assay or any method known in the art. As used herein, “Encapsulation” may refer to complete, substantial, or partial enclosure, containment, siege, or accommodation.

[0018] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., the heavy or light chain of an antibody) into an intact protein (e.g., an antibody), and / or post-translational modification of a polypeptide or a fully constructed protein (e.g., an antibody). In this disclosure, the terms “expression” and “production” and their grammatical equivalents are used interchangeably.

[0019] Functional: As used herein, a “functional” biomolecule is a biomolecule in which it exhibits the properties and / or activity that characterize it.

[0020] Half-life: As used herein, the term “half-life” refers to the time required for an amount of a nucleic acid or protein, such as its concentration or activity, to fall to half of its initial measured value over a given period.

[0021] Average particle size: As used herein, "average particle size" in relation to lipid nanoparticle compositions refers to the average diameter of the nanoparticle composition. In relation to dry powders in dry powder formulations, "average particle size" refers to the average diameter of the dry powders.

[0022] To improve, increase, or decrease: As used herein, the terms “improve,” “increase,” or “decrease,” or their grammatical synonyms, refer to relative values ​​to baseline measurements, such as measurements in the same individual before the initiation of the treatment described herein, or measurements in a control subject (or control subject) that has not received the treatment described herein. A “control subject” is a subject suffering from the same form of disease as the subject receiving treatment and being of approximately the same age as the subject receiving treatment.

[0023] Improved thermal stability: As used herein, the term “improved thermal stability” refers to the ability of a formulation (e.g., mRNA nanoparticles or reconstituted mRNA nanoparticles) to maintain its chemical structure and / or physical stability at high temperatures.

[0024] In some embodiments: When used herein, the phrase “in some embodiments,” or its grammatical equivalents such as “in a particular embodiment,” “in another embodiment,” and “in some other embodiment,” refers to all corresponding embodiments of the Disclosure unless the context clearly indicates otherwise.

[0025] In vitro: As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, or in a cell culture, rather than within a multicellular organism.

[0026] In vivo: As used herein, the term “in vivo” refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0027] Isolated: As used herein, the term “isolated” means (1) a substance and / or entity that has been separated from at least a portion of the components that accompanied it when it was first produced (whether in a natural and / or experimental setting), and / or (2) a substance and / or entity produced, prepared and / or manufactured by human hands. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components that accompanied them when they were first produced. In some embodiments, the isolated agent is pure to about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, excipients (e.g., buffers, solvents, water, etc.) should not be included in the calculation of the percentage purity of the isolated substance and / or entity.

[0028] Liquid control: As used herein, the terms “liquid control” or “liquid controls” refer to mRNA nanoparticle formulations that are formulated directly as liquid products, or reconstituted from, a dry powder formulation, rather than as a dry powder formulation. Thus, as used herein, a liquid control formulation is distinguished from a reconstituted formulation to which at least one solvent has been added to the mRNA-encapsulated dry powder formulation.

[0029] Reconstitute: As used herein, the terms “reconstitute” or “reconstituted” or related terms refer to the process of adding a liquid diluent to a dry component to produce a liquid formulation of a specific concentration.

[0030] Reconstituted preparations: As used herein, “reconstituted preparations” refers to the dissolution of a dry powder, lyophilized protein, spray-dried protein, or spray-dried nucleic acid (e.g., mRNA) or solvent-precipitated protein in a diluent. In some embodiments, “reconstituted preparations” refers to preparations prepared by dissolving or dispersing mRNA-nanoparticles in an aqueous solution for administration.

[0031] mRNA-LNP composition: As used herein, the term "mRNA-LNP composition" or its grammatical equivalent, such as "mRNA-LNP preparation," refers to a composition or preparation comprising one or more mRNA molecules encapsulated in LNPs.

[0032] Subject: As used herein, the term “subject” means a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse or primate) to which the provided composition (e.g., any of those described herein) may be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Humans include prenatal and postnatal forms. In many embodiments, the subject is human. The subject may be a patient, referring to a human being who visits a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may be a person suffering from or susceptible to a disease or disorder, the symptoms of the disease or disorder may or may not be present. Thus, the composition (e.g., any of those described herein) may be administered to a subject (e.g., a patient requiring the therapeutic and / or preventive treatment described herein), such as a patient (e.g., a human patient).

[0033] Sugars: As used herein, the term “sugar” includes monosaccharides, disaccharides, trisaccharides, and oligosaccharides comprising 1 to 10 monosaccharide units, e.g., 4 to 10 monosaccharide units. Any sugar is effective in formulating the Clostridium toxin pharmaceutical compositions disclosed herein, provided that a therapeutically effective amount of the Clostridium toxin active ingredient, most preferably botulinum toxin, is used with this sugar. It is assumed that it will be collected. In some embodiments, for example in lyophilized compositions, the sugar may function as a lyophilized protective agent. In some other embodiments, the sugar may function as an isotonic agent, for example in lyophilized formulations or liquid formulations. Monosaccharides are cyclic, such as polyhydroxyaldehydes or polyhydroxyketones having three or more carbon atoms, including aldoses, dialdos, aldoketoses, ketoses, and diketoses, and the parent monosaccharides have (potential) carbonyl groups, deoxy sugars, and amino sugars, as well as their derivatives. Monosaccharides include trioses such as glyceraldehyde and dihydroxyacetone; tetroses such as erythrose, erythrulose, and threose; pentoses such as arabinose, lyxose, ribose, ribulose, xylose, and xylulose; glucose hexoses such as allose, altrose, fructose, fucose, galactose, sucrose, and mannoheptulose; octose such as octulose and 2-keto-3-deoxy-mannooctone; groose, idose, mannose, psicose, rhamnose, sorbose, tagatose, talose, and trehalose; nonoses such as sialose; and decorose. Oligosaccharides are compounds in which at least two types of monosaccharide units are linked by glycosidic bonds. Depending on the number of units, they are called disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, xusaccharides, decasaccharides, etc. Oligosaccharides can be unbranched, branched, or cyclic. Common disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, genthiobiose, kojierbiose, laminaribiose, mannobiose, melibiose, nigerose, rutinose, and xylobiose.Common trisaccharides include, but are not limited to, raffinose, acarbose, maltotriose, and meletitol. Other non-exclusive examples of special-purpose sugar excipients are described, for example, in Ansel (1999), Gennaro (2000), Hardman (2001), and Rowe (2003), each of which is incorporated by reference.

[0034] Sugar alcohols: As used herein, the term “polyalcohol” is synonymous with “sugar alcohol,” “polyhydric alcohol,” and “polyol,” and is characterized by an alcohol group (CH2OH) instead of an aldehyde group (CHO). Examples include mannose derived from mannose, xylitol derived from xylose, and lactitol derived from lactulose. Non-limiting examples of polyols include glycols, glycerols, arabitol, erythritol, xylitol, maltitol, sorbitol (glucitol), mannitol, inositol, lactitol, galactitol (iditol), and isomalt. Other non-limiting examples of sugar excipients are described, for example, in Ansel (1999), Gennaro (2000), Hardman (2001), and Rowe (2003), each of which is incorporated by reference in whole.

[0035] This application provides, in particular, a dried powder formulation of mRNA-LNP that offers unexpectedly excellent properties, including a heat-stable dried powder product (DPP) and a reconstituteable dried powder product (reconstituteability and heat stability).

[0036] The terms "dried powder product" (DPP) and "dried powder" (DP) may be used interchangeably with the term "dried powder formulation" as used herein.

[0037] For example, the dry powder formulations described herein may be suitable for use as dry powder products without reconstitution, as well as for use after reconstitution. The ability to reconstitute the dry powder formulations described herein is particularly beneficial for use in various delivery modes, including but not limited to parenteral (intramuscular, intravenous, subcutaneous, etc.) and mucosal (oral, sublingual, intranasal, etc.) delivery. Surprisingly, the inventors of this disclosure have found that desired important attributes of the reconstituted product of the dry powder formulations described herein can be achieved by using the excipients described herein and the weight ratio of the excipients to the total lipid content in the DPP in a spray-drying process.

[0038] As disclosed herein, “reconstituteable dry powder formulations” and “dry powder formulations for reconstitution” may be used interchangeably.

[0039] Various exemplary aspects and embodiments of this disclosure are described in detail in the following sections. The use of these sections is not intended to limit this disclosure. Each section may be applied to any aspect of this disclosure. In this application, the use of “or” means “and / or” unless otherwise stated.

[0040] In some embodiments, the disclosure features a dry powder formulation comprising messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs), the lipid nanoparticles comprising one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the formulation comprises a sugar or sugar alcohol, and the weight (w / w) ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticles is at least about 5. In some embodiments, the dry powder formulation may be reconstituted for parenteral administration by reconstitution with water or a buffer. The reconstituted form of the dry powder formulation may be referred to as a “reconstituted dry powder formulation.” In some embodiments, the dry powder formulation comprises a dry powder having a particle size between 18 μm or about 1 μm to about 8 μm (e.g., average particle size).

[0041] In another embodiment, the present disclosure features a method for preparing a dry powder formulation as described herein. In some embodiments, the method comprises providing a first mixture containing lipid nanoparticles (i.e., mRNA-LNPs) encapsulating mRNA; adding an excipient selected from sugars or sugar alcohols (e.g., trehalose) to the mRNA-LNPs at a concentration of 2–10% (w / w) to obtain a second mixture; and spray-drying the second mixture. In some embodiments, a dry powder formulation having a particle size between 18 μm is obtained, for example, the dry powder in the formulation having an average particle size of about 1 μm to about 8 μm. In some embodiments, the lipid nanoparticles used in the method of the present disclosure include one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the dry powder formulation can be reconstituted with water or buffer for parenteral administration. In some embodiments, the dry powder containing the excipient is stable for at least one year (e.g., after storage at 2–8°C for at least one year).

[0042] Dried powder preparation containing sugar or sugar alcohol This disclosure provides a stable dry powder formulation containing mRNA-loaded lipid nanoparticles (mRNA-LNPs) for therapeutic use. The dry powder formulation of this disclosure is a heat-stable and / or reconstituted dry powder formulation.

[0043] In some embodiments, the dry powder formulations of the Disclosure include excipients for enhancing the production of a heat-stable and / or reconstituted dry powder formulation. In some embodiments, the dry powder formulations of the Disclosure include excipients for enhancing the production of a reconstituted dry powder formulation useful for parenteral administration. In some embodiments, the dry powder formulations described herein include sugars and / or sugar alcohols (e.g., trehalose or sucrose) as excipients. In some embodiments, the dry powder formulations described herein include sugars as excipients. In some embodiments, the dry powder formulations described herein include sugar alcohols as excipients.

[0044] In some embodiments, the sugar is selected from glucose, fructose, mannose, galactose, mannitol, sorbitol, lactose, trehalose, sucrose, xylose, ribulose, maltose, tagatose, galactose, rhamnose, ribulose, threose, arabinose, xylose, lyxose, allose, altrose, idose, palatinose, reduced isomalt-oligosaccharide, reduced xylo-oligosaccharide, cellobiose, trehalose, raffinose, starch, dextran, maltodextrin, cyclodextrin, inulin, or any combination thereof. In some embodiments, the sugar alcohol is selected from erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, propylene glycol, glycerol (glycerin), treitol, galactitol, adonitol, dalcitol, pentaerythritol, or any combination thereof.

[0045] In some embodiments, the sugar or sugar alcohol is selected from trehalose, mannitol, lactose, xylitol, and sucrose, and any combination thereof. In some embodiments, the sugar is trehalose. In some embodiments, the sugar or sugar alcohol is mannitol. In some embodiments, the sugar is lactose. In some embodiments, the sugar or sugar alcohol is xylitol. In some embodiments, the sugar or sugar alcohol is sucrose.

[0046] Sugars or sugar alcohols and total lipids The ratio of excipients to total lipid content in a dry powder formulation can provide a reconstituted dry powder with optimal properties. In some embodiments, the weight ratio (w / w) of sugar or sugar alcohol (e.g., sucrose or trehalose) to total lipids in lipid nanoparticles provides optimal stability for the reconstituted dry powder formulation.

[0047] In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in LNP is about 2 to 10, about 4 to 8, or about 4 to 6. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in LNP is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in LNP is 2 to 10. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in LNP is 4 to 8. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in LNP is 4 to 6.

[0048] In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 2. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 3. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 4. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 5. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 6. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 7. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 8. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 9. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 10. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 11. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 12. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 13. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 14. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 15. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 16. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 17. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 18.In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 19. In some embodiments, the w / w ratio of sugar or sugar alcohol to total lipids in lipid nanoparticles is at least about 20.

[0049] In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.1. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.2. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.3. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.4. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.5. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.6. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.7. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 5.8. In some embodiments, the dry powder formulation includes having a w / w ratio of excipients to total lipids of at least 5.9.

[0050] In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.1. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.2. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.3. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.4. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.5. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.6. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.7. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 11.8. In some embodiments, the dry powder formulation includes having a w / w ratio of excipients to total lipids of at least 11.9.

[0051] In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.0. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.1. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.2. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.3. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.4. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.5. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.6. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.7. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.8. In some embodiments, the dry powder formulation includes a w / w ratio of excipient to total lipids of at least 15.9.

[0052] In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.1. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.2. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.3. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.4. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.5. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.6. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.7. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.8. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 5.9.

[0053] In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.1. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.2. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.3. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.4. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.5. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.6. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.7. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.8. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 11.9.

[0054] In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.0. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.1. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.2. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.3. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.4. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.5. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.6. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.7. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.8. In some embodiments, the w / w ratio of the excipient to the total lipids in the LNP is at least 15.9.

[0055] In some embodiments, the sugar or sugar alcohol is selected from trehalose, mannitol, lactose, xylitol, and sucrose, as well as any combination thereof.

[0056] Exemplary features of the dried powder formulation described herein In some embodiments, the dry powder formulations containing sugars or sugar alcohols disclosed herein (e.g., reconstituteable dry powder formulations) have desired properties, including, but not limited to, better shelf life, efficacy, thermal stability, tissue absorption, and / or encapsulation efficacy. Exemplary, non-limiting beneficial features are described herein.

[0057] a.Save For example, the dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in terms of storage and / or storage stability.

[0058] In some embodiments, the dry powder formulations described herein may have improved shelf life (e.g., including storage at relatively high temperatures that do not require extremely low temperatures). In some embodiments, the reconstituteable dry powder formulations described herein are also characterized by their improved storage properties. For example, in certain embodiments, reconstituteable dry powder formulations may be stored under refrigeration and may remain stable for extended periods (e.g., as demonstrated by minimal or no loss of their intended pharmaceutical or biological activity) (e.g., stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months (including any value and partial ranges between them) or longer when stored at temperatures such as about 2–8°C (e.g., 4°C) or -20°C as described herein). In other embodiments, the reconstituteable dry powder formulations described herein may be stored without refrigeration and may remain stable for extended periods (for example, stable for at least about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months (including any values ​​and partial ranges between them) or longer when stored at ambient temperatures including about 25°C).

[0059] In some embodiments, the reconfigurable dry powder formulations described herein provide equivalent shelf life compared to equivalent non-dried powder formulations. In some embodiments, the shelf life does not change significantly during storage of the dry powder formulation, including any exemplary period and / or temperature described herein. In some embodiments, the shelf life of the dry powder formulations described herein does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between) after storage, including any exemplary period and / or temperature described herein.

[0060] In some embodiments, the reconstituteable dry powder formulations described herein provide a comparable shelf life to the dry powder formulation before reconstitution. In some embodiments, the shelf life of the reconstituted dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between).

[0061] b. Stability For example, the reconstituteable dry powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects on the stability of the formulations.

[0062] In some embodiments, the reconstituteable dry powder formulations containing sugars or sugar alcohols described herein are stable for at least one week. In some embodiments, the reconstituteable dry powder formulations are stable for at least two weeks. In some embodiments, the reconstituteable dry powder formulations are stable for at least three weeks. In some embodiments, the reconstituteable dry powder formulations are stable for at least four weeks. In some embodiments, the reconstituteable dry powder formulations are stable for at least five weeks. In some embodiments, the reconstituteable dry powder formulations are stable for at least one month. In some embodiments, the reconstituteable dry powder formulations are stable for at least two months. In some embodiments, the reconstituteable dry powder formulations are stable for at least three months. In some embodiments, the reconstituteable dry powder formulations are stable for at least four months. In some embodiments, the reconstituteable dry powder formulations are stable for at least five months. In some embodiments, the reconstituteable dry powder formulations are stable for at least six months. In some embodiments, the reconstituteable dry powder formulations are stable for at least seven months. In some embodiments, the reconstituteable dry powder formulations are stable for at least eight months. In some embodiments, the reconstituteable dry powder formulations are stable for at least nine months. In some embodiments, the reconstituted dry powder formulation is stable for at least 10 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 11 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 1 year. In some embodiments, the reconstituted dry powder formulation is stable for about 1 year or more.

[0063] In some embodiments, the stability of a reconstituted dry powder formulation containing a sugar or sugar alcohol described herein increases by at least about one week compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least two weeks compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least three weeks compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least four weeks compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least about five weeks compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder increases by at least one month compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder increases by at least two months compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least three months compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder increases by at least four months compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder increases by at least five months compared to a liquid control. In some embodiments, the stability of a reconstituted dry powder formulation increases by at least six months compared to a liquid control. In some embodiments, the stability of the reconstituted dry powder increases by at least 7 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder increases by at least 8 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least 9 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder increases by at least 10 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder increases by at least 11 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least 1 year compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by more than 1 year compared to the liquid control.

[0064] In some embodiments, the reconstituteable dry powder formulations comprising sugars or sugar alcohols described herein exhibit improved thermal stability compared to liquid controls. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least one week. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least two weeks. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least three weeks. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least four weeks. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least about five weeks. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for one month. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least two months. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least three months. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least four months. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least five months. In some embodiments, the reconstituteable dry powder formulations are stable at 4°C for at least six months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 7 months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 8 months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 9 months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 10 months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 11 months. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least 1 year. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for more than 1 year.

[0065] In some embodiments, the reconstituted dry powder formulations containing sugars or sugar alcohols described herein are stable at 25°C for at least one week. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least two weeks. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least three weeks. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least four weeks. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least about five weeks. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for one month. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least two months. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least three months. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least four months. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least five months. In some embodiments, the reconstituted dry powder formulations are stable at 25°C for at least six months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 7 months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 8 months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 9 months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 10 months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 11 months. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 1 year. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for more than 1 year.

[0066] In some embodiments, the reconstituteable dry powder formulations containing sugars or sugar alcohols described herein are stable at -20°C for at least one week. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least two weeks. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least three weeks. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least four weeks. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least five weeks. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for one month. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least two months. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least three months. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least four months. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least five months. In some embodiments, the reconstituteable dry powder formulations are stable at -20°C for at least six months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 7 months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 8 months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 9 months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 10 months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 11 months. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 1 year. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for more than 1 year.

[0067] In certain embodiments, the reconstituteable dried powder formulations described herein demonstrate a negligible reduction in pharmacological or biological activity (e.g., a reduction in the biological or pharmacological activity of the encapsulated polynucleotides of less than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (including all values ​​and ranges between them)) (e.g., when compared to a previous dried powder formulation or a control formulation without spray drying or a dried powder formulation before storage).

[0068] In some embodiments, the reconfigurable dry powder formulations described herein offer comparable stability to equivalent non-dried powder formulations. In some embodiments, the stability does not change significantly during storage of the dry powder formulation, including any exemplary storage, duration, and / or temperature described herein. In some embodiments, the stability of the dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (including all values ​​and ranges between them) (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) after storage, including any exemplary storage, duration, and / or temperature described herein.

[0069] In some embodiments, the reconstituteable dry powder formulations described herein provide comparable stability to the dry powder formulation before reconstitution. In some embodiments, the stability of the reconstituteable dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between).

[0070] c.mRNA integrity For example, the dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in maintaining the integrity of mRNA encapsulated within LNPs.

[0071] In some embodiments, mRNA maintains 60% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 61% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 62% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 63% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 64% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 65% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 66% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 67% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 68% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 69% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between).In some embodiments, mRNA maintains 70% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 71% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 72% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 73% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 74% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 75% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 76% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 77% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 78% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 79% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between).In some embodiments, mRNA maintains 80% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 81% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 82% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 83% or more integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 84% ​​or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 85% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 86% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 87% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 88% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between). In some embodiments, mRNA maintains 89% or greater integrity after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer (including all values ​​and ranges in between).In some embodiments, mRNA maintains 90% or more integrity for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer.

[0072] In some embodiments, mRNA maintains 60% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 61% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 62% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 63% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 64% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 65% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 72% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 73% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 74% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 75% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 76% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains more than 78% integrity after being stored at 4°C for at least 6 months.In some embodiments, mRNA maintains 79% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 88% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 89% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 90% or more integrity after being stored at 4°C for at least 6 months.

[0073] In some embodiments, mRNA maintains 60% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 61% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 62% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 63% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 64% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 65% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 72% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 73% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 74% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 75% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 76% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at 25°C for at least 6 months.In some embodiments, mRNA maintains 78% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 79% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 88% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 89% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at 25°C for at least 6 months.

[0074] In some embodiments, mRNA maintains 60% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 61% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 62% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 63% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 64% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 65% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 72% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 73% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 74% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 75% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 76% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at -20°C for at least 6 months.In some embodiments, mRNA maintains 78% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 79% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 88% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 89% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at -20°C for at least 6 months.

[0075] In some embodiments, the mRNA retains at least 60% integrity, for example, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (including any values ​​and subranges between them) after being stored at 2–8°C (e.g., about 4°C) for at least about one year.

[0076] In some embodiments, mRNA maintains at least 70% integrity after being stored at 2–8°C for at least about one year. In some embodiments, mRNA maintains at least 75% integrity after being stored at 2–8°C for at least about one year. In some embodiments, mRNA maintains at least 80% integrity after being stored at 2–8°C for at least about one year. In some embodiments, mRNA maintains at least 85% integrity after being stored at 2–8°C for at least about one year. In some embodiments, mRNA maintains at least 90% integrity after being stored at 2–8°C for at least about one year. In some embodiments, mRNA maintains at least 95% integrity after being stored at 2–8°C for at least about one year.

[0077] In some embodiments, mRNA maintains at least 70% integrity after being stored at 4°C for at least about one year. In some embodiments, mRNA maintains at least 75% integrity after being stored at 4°C for at least about one year. In some embodiments, mRNA maintains at least 80% integrity after being stored at 4°C for at least about one year. In some embodiments, mRNA maintains at least 85% integrity after being stored at 4°C for at least about one year. In some embodiments, mRNA maintains at least 90% integrity after being stored at 4°C for at least about one year. In some embodiments, mRNA maintains at least 95% integrity after being stored at 4°C for at least about one year.

[0078] In some embodiments, the reconstituteable dry powder formulations described herein provide comparable mRNA integrity to equivalent non-dried powder formulations. In some embodiments, mRNA integrity does not change significantly during storage of the reconstituteable dry powder formulation, including any exemplary storage, duration, and / or temperature described herein. In some embodiments, the mRNA integrity of the reconstituteable dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between) after storage, including by any exemplary storage, duration, and / or temperature described herein.

[0079] In some embodiments, the reconstituted dry powder formulations described herein provide equivalent mRNA integrity compared to the dry powder formulation before reconstitution. In some embodiments, the mRNA integrity of the reconstituted dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between) after reconstitution, including by any of the methods described herein.

[0080] d.Particle size For example, the reconstituteable dry powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects on the particle size of LNPs in the formulation, for example, in maintaining the particle size of LNPs (e.g., average particle size).

[0081] In some embodiments, the reconstituteable dry powder formulations described herein include dry powder having a particle size (e.g., average particle size) of 0.5 to 10 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is 1 to 5 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is 1 to 4 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is 1 to 3 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is 1 to 2 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 1 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 2 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 3 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 4 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 5 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 6 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 7 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 8 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 9 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder in the formulation is about 10 μm.

[0082] In some embodiments, the particle size (e.g., average particle size) of the dry powder in the reconstituted dry powder formulations disclosed herein is desirable for delivery to various tissues. For example, dry powder formulations containing a particle size of about 5 microns or less (e.g., average particle size) are particularly useful for pulmonary delivery. In an additional example, dry powder formulations containing trehalose have been found to have a particle size of 5 microns or less and may be suitable for pulmonary delivery.

[0083] In some embodiments, the reconstituteable dry powder formulations described herein provide equivalent particle size (e.g., average particle size) compared to equivalent non-dried powder formulations. In some embodiments, the particle size (e.g., average particle size) does not change significantly during storage of the dry powder formulation, including any exemplary storage, duration, and / or temperature described herein. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between) after storage, including by any exemplary storage, duration, and / or temperature described herein.

[0084] In some embodiments, the reconstituteable dry powder formulations described herein provide equivalent particle size (e.g., average particle size) compared to the dry powder formulation before reconstitution. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between).

[0085] e. Delivery route The reconstituted dry powder formulations described herein may be useful in a variety of delivery routes. Exemplary embodiments are described herein.

[0086] In some embodiments, reconstituteable dry powder formulations containing sugars or sugar alcohols as described herein enable the administration of mRNA-nanoparticles as a liquid formulation in desired doses. In some embodiments, reconstituteable dry powder formulations containing dry powders having dimensions of 6 microns or less, 5 microns or less, 4 microns or less, 3 microns or less, 2 microns or less, and 1 micron or less (including all values ​​and ranges in between) may be suitable for pulmonary delivery. Pharmaceutical compositions containing the reconstituted dry powder formulations described herein may be administered subconjunctivally, sublingually, to subjects requiring it by any one of several routes that effectively deliver an effective amount of the compound. Non-limiting examples of preferred routes of administration include topical, oral, nasal, intrathecal, enteral, oral cavity, sublingual, transdermal, rectal, vaginal, intraocular, and parenteral administration (subcutaneous, intravenous, intramuscular, intrasternal, intracavernosal, intracavitary, and intraurethral injection and / or infusion).

[0087] In some embodiments, the reconstituted dry powder formulation is suitable for mucosal delivery. In some embodiments, the reconstituted dry powder formulation is suitable for oral delivery. In some embodiments, the reconstituted dry powder formulation is suitable for sublingual delivery. In some embodiments, the reconstituted dry powder formulation is suitable for intranasal delivery. In some embodiments, the reconstituted dry powder formulation is suitable for oral delivery.

[0088] f. Improved delivery efficacy The reconstituted dry powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in the delivery of formulations to target tissues.

[0089] In some embodiments, the reconstituteable dry powder formulations described herein are useful for improving therapeutic efficacy, including improved delivery of mRNA to cells.

[0090] In some embodiments, reconstituteable dry powder formulations have improved delivery of mRNA to various tissues compared to liquid controls. In some embodiments, the formulations described herein have improved delivery when used orally, mucosally, intravenously, intramuscularly, subcutaneously, transdermally, or orally. In some embodiments, the formulations described herein have improved delivery when used in a delivery route preferred to the disclosure.

[0091] In some embodiments, the reconstituteable dry powder formulations described herein provide equivalent delivery efficacy compared to equivalent non-dried powder formulations. In some embodiments, the delivery efficacy does not change significantly during storage of the reconstituteable dry powder formulation, including the exemplary storage, duration, and / or temperature described herein. In some embodiments, the delivery efficacy of the dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between) after storage, including by any of the exemplary storage, duration, and / or temperature described herein.

[0092] In some embodiments, the reconstituteable dry powder formulations described herein provide equivalent delivery efficacy compared to the dry powder formulation before reconstitution. In some embodiments, the delivery efficacy of the reconstituteable dry powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5%) (including all values ​​and ranges in between).

[0093] g. Sugar-to-lipid ratio The reconstituted dry powder formulations described herein may have an optimal particle size for tissue-specific delivery.

[0094] In some embodiments, a reconstituteable dry powder formulation containing trehalose is used for lung delivery. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 1. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 2. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 3. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 4. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 5. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 6. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 7. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 8. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 9. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 10. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 11. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 12. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 13. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 14. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 15. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 16. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 17. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 18. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 19. In some embodiments, the ratio of trehalose concentration to total lipid concentration is greater than 20.

[0095] h. Encapsulation efficiency The reconstituted dry powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in the encapsulation efficiency of LNPs in formulations.

[0096] In some embodiments, the reconstituteable dry powder formulations containing sugars or sugar alcohols described herein exhibit higher encapsulation efficiency compared to a liquid control or a dry powder without sugars or sugar alcohols. In some embodiments, the encapsulation efficiency exceeds 60%. In some embodiments, the encapsulation efficiency exceeds 61%. In some embodiments, the encapsulation efficiency exceeds 62%. In some embodiments, the encapsulation efficiency exceeds 63%. In some embodiments, the encapsulation efficiency exceeds 64%. In some embodiments, the encapsulation efficiency exceeds 65%. In some embodiments, the encapsulation efficiency exceeds 66%. In some embodiments, the encapsulation efficiency exceeds 67%. In some embodiments, the encapsulation efficiency exceeds 68%. In some embodiments, the encapsulation efficiency exceeds 69%. In some embodiments, the encapsulation efficiency exceeds 70%. In some embodiments, the encapsulation efficiency exceeds 71%. In some embodiments, the encapsulation efficiency exceeds 72%. In some embodiments, the encapsulation efficiency exceeds 73%. In some embodiments, the encapsulation efficiency exceeds 74%. In some embodiments, the encapsulation efficiency exceeds 75%. In some embodiments, the encapsulation efficiency exceeds 76%. In some embodiments, the encapsulation efficiency exceeds 77%. In some embodiments, the encapsulation efficiency exceeds 78%. In some embodiments, the encapsulation efficiency exceeds 79%. In some embodiments, the encapsulation efficiency exceeds 80%. In some embodiments, the encapsulation efficiency exceeds 81%. In some embodiments, the encapsulation efficiency exceeds 82%. In some embodiments, the encapsulation efficiency exceeds 83%. In some embodiments, the encapsulation efficiency exceeds 84%. In some embodiments, the encapsulation efficiency exceeds 85%. In some embodiments, the encapsulation efficiency exceeds 86%. In some embodiments, the encapsulation efficiency exceeds 87%. In some embodiments, the encapsulation efficiency exceeds 88%. In some embodiments, the encapsulation efficiency exceeds 89%. In some embodiments, the encapsulation efficiency exceeds 90%. In some embodiments, the encapsulation efficiency exceeds 91%. In some embodiments, the encapsulation efficiency exceeds 92%. In some embodiments, the encapsulation efficiency exceeds 93%.In some embodiments, the encapsulation efficiency exceeds 94%. In some embodiments, the encapsulation efficiency exceeds 95%. In some embodiments, the encapsulation efficiency exceeds 96%. In some embodiments, the encapsulation efficiency exceeds 97%. In some embodiments, the encapsulation efficiency exceeds 98%. In some embodiments, the encapsulation efficiency exceeds 99%.

[0097] In some embodiments, the particle size (e.g., average particle size) of the dry powder contained in the reconstituteable dry powder formulation containing sugar or sugar alcohol described herein is less than 3 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 4 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 5 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 6 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 7 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 8 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 9 μm. In some embodiments, the particle size (e.g., average particle size) of the reconstituteable dry powder is less than 10 μm.

[0098] In some embodiments, the reconstituteable dry powder formulations described herein exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally involve contacting one or more target cells with, for example, the dry powder formulation described herein, so that the one or more target cells are transfected with a substance encapsulated therein (e.g., one or more polynucleotides or mRNA). For example, as also further described herein, the dry powder formulations may be beneficial for needle-free (i.e., non-invasive) administration routes, such as delivery via a mucosal surface.

[0099] Exemplary characteristics of the reconstituted dried powder formulation described herein The reconstituteable dried powder formulations described herein provide beneficial properties after reconstitution to desired mRNA-LNP formulations, including, but not limited to, the exemplary beneficial properties described herein.

[0100] In some embodiments, the reconstituteable dry powder formulations described herein can retain desirable characteristics after reconstitution, including, but not limited to, beneficial stability (e.g., determined with respect to the particle size (e.g., average particle size) of the reconstituted lipid nanoparticles containing such compositions). In some embodiments, reconstitution of the dry powder formulations described herein does not significantly alter or change the particle size (e.g., average particle size) and / or encapsulation efficiency of the reconstituted lipid nanoparticles. In some embodiments, reconstitution of the dry powder formulations described herein does not significantly alter or change the therapeutic efficacy (e.g., mRNA integrity and / or activity) of the reconstituted mRNA.

[0101] a. Lack of aggregation For example, the reconstituted dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in stabilizing the formulation by minimizing aggregation.

[0102] In some embodiments, disclosed herein are dry powder compositions in which lipid nanoparticles do not flocculate or aggregate upon reconstitution (e.g., according to the methods described herein), or alternatively, limited or negligible flocculation or aggregation (e.g., determined by the particle size of the reconstituted lipid nanoparticles (e.g., average particle size)) are demonstrated. Accordingly, in some embodiments, upon reconstitution of the dry powder formulations described herein, the lipid nanoparticles have a geometric particle size distribution Dv50 of less than about 500 nm (e.g., less than about 300 nm, 200 nm, 150 nm, 125 nm, 120 nm, 100 nm, 75 nm, 50 nm, less than 25 nm, or less (including all values ​​and ranges in between)). Similarly, in some embodiments, when the dried powder formulations described herein are reconstituted, the lipid nanoparticles have a geometric particle size distribution Dv90 of less than about 750 nm (e.g., less than about 700 nm, 500 nm, 300 nm, 200 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or less (including all values ​​and ranges in between)).

[0103] In some embodiments, upon reconstitution with a suitable rehydration medium, the reconstituted dry powder formulation exhibits pharmacological or biological activity equivalent to that observed in the previous dry powder formulation. For example, in some embodiments, the pharmacological or biological activity of the encapsulated polynucleotides in the reconstituted formulation is equivalent to that observed in the dry powder formulation of the previous composition, or, instead, exhibits a negligible reduction in pharmacological or biological activity (e.g., a reduction in the biological or pharmacological activity of the encapsulated polynucleotides of less than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, 50% (including all values ​​and ranges between them)).

[0104] Methods for transfecting one or more target cells are also provided herein. In some embodiments, the reconfigured compositions of the Disclosure (e.g., lipid nanoparticles) exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Such methods generally involve contacting one or more target cells with a reconfigured dried powder formulation described herein so that the one or more target cells are transfected with a substance encapsulated therein (e.g., one or more polynucleotides or mRNAs).

[0105] b. Reconstituted solvent In some embodiments, the reconstituteable dry powder formulations disclosed herein, comprising mRNA nanoparticles and excipients, are reconstituted with a solvent. Reconstitution of the dry powder product offers advantages for parenteral administration.

[0106] In some embodiments, the solvent is water.

[0107] In some embodiments, the solvent is a buffer. The buffer or pH adjuster in the emulsion composition is used to adjust the pH to a desired range. Exemplary buffers include, but are not limited to, phosphate buffer, citrate buffer, Tris buffer, carbonate buffer, succinate buffer, maleate buffer, and borate buffer. In some embodiments, the buffer is selected from the group consisting of phosphate-buffered saline (PBS), modified PBS, and citrate buffer. In some embodiments, the buffer may be any buffer suitable for the formulations and methods described herein.

[0108] In some embodiments, the reconstitution of the dry powder product involves direct reconstitution in a buffer. In some embodiments, the reconstitution of the dry powder product in a buffer includes (1) a step of reconstitution with water and (2) a step of reconstitution with a buffer.

[0109] c. Stability The reconstituted dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects on formulation stability.

[0110] In some embodiments, the reconstituted dry powder formulations containing sugars or sugar alcohols described herein are stable for at least one week. In some embodiments, the reconstituted dry powder formulations are stable for at least two weeks. In some embodiments, the reconstituted dry powder formulations are stable for at least three weeks. In some embodiments, the reconstituted dry powder formulations are stable for at least four weeks. In some embodiments, the reconstituted dry powder formulations are stable for at least one month. In some embodiments, the reconstituted dry powder formulations are stable for at least two months. In some embodiments, the reconstituted dry powder formulations are stable for at least three months. In some embodiments, the reconstituted dry powder formulations are stable for at least four months. In some embodiments, the reconstituted dry powder formulations are stable for at least five months. In some embodiments, the reconstituted dry powder formulations are stable for at least six months. In some embodiments, the reconstituted dry powder formulations are stable for at least seven months. In some embodiments, the reconstituted dry powder formulations are stable for at least eight months. In some embodiments, the reconstituted dry powder formulations are stable for at least nine months. In some embodiments, the reconstituted dry powder formulations are stable for at least ten months. In some embodiments, the reconstituted dry powder formulations are stable for at least eleven months. In some embodiments, the reconstituted dried powder formulation is stable for at least one year. In some embodiments, the reconstituted dried powder formulation is stable for about one year or more.

[0111] In some embodiments, the stability of the reconstituted dry powder formulation containing sugar or sugar alcohol increases by at least one week compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least two weeks compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least three weeks compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least four weeks compared to the liquid control. In some embodiments, the stability of the dry powder increases by at least one month compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least two months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least three months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least four months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least five months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least six months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least seven months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least eight months compared to the liquid control. The stability of the reconstituted dry powder formulation increases by at least 9 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least 10 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least 11 months compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by at least 1 year compared to the liquid control. In some embodiments, the stability of the reconstituted dry powder formulation increases by more than 1 year compared to the liquid control.

[0112] In some embodiments, the reconstituted dry powder formulation containing sugar or sugar alcohol exhibits better thermal stability compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least one week compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 4°C for at least two weeks. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least three weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least four weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for one month compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least two months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least three months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least four months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least five months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least six months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 7 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 8 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 9 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 10 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 11 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for at least 1 year compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is thermally stable at 4°C for more than 1 year compared to a liquid control.

[0113] In some embodiments, the reconstituted dry powder formulation containing sugar or sugar alcohol is stable at 25°C for at least one week compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least two weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least three weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least four weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for one month compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least two months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least three months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least four months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least five months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least six months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 7 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 8 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 9 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 10 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 11 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 1 year. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for more than 1 year.

[0114] In some embodiments, the reconstituted dry powder formulation containing sugar or sugar alcohol is stable at -20°C for at least one week compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least two weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least three weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least four weeks compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for one month compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least two months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least three months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least four months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least five months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least six months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 7 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 8 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 9 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 10 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 11 months compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 1 year compared to a liquid control. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for more than 1 year compared to a liquid control.

[0115] In some embodiments, reconstituted dried powder formulations have demonstrated negligible reductions in pharmacological or biological activity (e.g., reductions in the biological or pharmacological activity of encapsulated polynucleotides of less than approximately 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, and 50% (including all values ​​and ranges in between)) compared to previous dried powder formulations.

[0116] In some embodiments, the reconstituted dried powder formulations described herein offer comparable stability to equivalent non-dried powder formulations. In some embodiments, the stability does not change significantly during storage of the dried powder formulation, including by any of the exemplary storage, duration, and / or temperatures described herein. In some embodiments, the stability of the reconstituted dried powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (including all values ​​and ranges between them) (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5% (including all values ​​and ranges between them)) after storage, including by any of the exemplary storage, duration, and / or temperatures described herein.

[0117] In some embodiments, the reconstituted dry powder formulations described herein provide comparable stability to the reconstituted dry powder formulation before reconstitution. In some embodiments, the stability of the reconstituted dry powder formulation does not change beyond about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, 50% (including all values ​​and ranges between them) (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5% (including all values ​​and ranges between them)) after reconstitution, including by any of the methods described herein.

[0118] d.Save The reconstituted dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects in terms of storage and / or storage stability.

[0119] In some embodiments, the dry powder formulations described herein may have improved shelf life (e.g., including storage at relatively high temperatures that do not require extremely low temperatures). In some embodiments, the reconstituted dry powder formulations described herein are also characterized by their improved storage properties. For example, in some embodiments, the reconstituted dry powder formulations may be stored under refrigeration and remain stable for long periods (e.g., as demonstrated by minimal or no loss of their intended pharmaceutical or biological activity) (e.g., stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer when stored at temperatures such as those described herein, including about 4°C or -20°C). In some embodiments, the reconstituteable dry powder formulation remains stable for extended periods without refrigeration (for example, stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer when stored at ambient temperatures including about 25°C).

[0120] In some embodiments, the reconstituted dried powder formulations described herein provide an equivalent shelf life compared to an equivalent non-dried powder formulation. In some embodiments, the shelf life does not change significantly during storage of the reconstituted dried powder formulation, including any exemplary storage, duration, and / or temperature described herein. In some embodiments, the shelf life of the reconstituted dried powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, and 50% (including all values ​​and ranges between them) after storage, including any exemplary storage, duration, and / or temperature described herein (for example, less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5% (including all values ​​and ranges between them)).

[0121] In some embodiments, the reconstituted dried powder formulations described herein provide a shelf life equivalent to that of the dried powder formulation before reconstitution. In some embodiments, the shelf life of the reconstituted dried powder formulation does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, or 50% (including all values ​​and ranges between them) after reconstitution, including by any of the methods described herein.

[0122] e. Particle size The reconstituted dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects on the particle size (e.g., average particle size) of the LNPs of the formulation.

[0123] In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 80 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 81 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 82 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 83 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 84 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 85 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 86 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 87 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 88 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 89 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 90 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 91 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 92 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 93 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 94 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 95 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 96 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 97 nm.In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 98 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 99 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 100 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 101 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 102 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 103 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 104 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 105 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 106 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 107 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 108 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 109 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 110 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 111 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 112 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 113 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 114 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 115 nm.In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 116 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 117 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 118 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 119 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 120 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 121 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 122 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 123 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 124 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 125 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 126 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 127 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 128 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 129 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 130 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 131 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 132 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 133 nm.In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 134 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 135 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 136 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 137 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 138 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 139 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 140 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 141 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 142 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 143 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 144 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 145 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 146 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 147 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 148 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 149 nm. In some embodiments, the particle size of the reconstituted dry powder formulation containing sugar or sugar alcohol as an excipient is less than 150 nm.

[0124] In some embodiments, the “particle size of the reconstituted dry powder formulation” as described herein refers to the average particle size of LNPs in the reconstituted dry powder formulation. Therefore, in some embodiments, the average particle size of LNPs in a reconstituted dry powder formulation containing sugars or sugar alcohols as excipients is less than 150 nm, for example less than 145 nm, less than 140 nm, less than 135 nm, less than 130 nm, less than 125 nm, less than 120 nm, less than 115 nm, less than 110 nm, less than 105 nm, less than 100 nm, less than 95 nm, less than 90 nm, less than 85 nm, or less than 80 nm (including all values ​​and ranges in between).

[0125] In some embodiments, the reconstituted dried powder formulations described herein provide equivalent particle size (e.g., average particle size of LNPs in the reconstituted formulation) compared to equivalent non-dried powder formulations. In some embodiments, the particle size (e.g., average particle size of reconstituted LNPs) does not change significantly during storage of the reconstituted dried powder formulation, including any exemplary storage, duration, and / or temperature described herein. In some embodiments, the particle size of the reconstituted dried powder formulation (e.g., average particle size of LNPs in the reconstituted formulation) does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, 50% (including all values ​​and ranges between them) (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5% (including all values ​​and ranges between them)) after storage, including any exemplary storage, duration, and / or temperature described herein.

[0126] In some embodiments, the reconstituted dried powder formulations described herein provide equivalent particle size (e.g., average particle size of LNPs in the reconstituted formulation) compared to the reconstituted dried powder formulation before reconstitution. In some embodiments, the particle size of the reconstituted dried powder formulation (e.g., average particle size of LNPs in the reconstituted formulation) does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, 50% (including all values ​​and ranges between them) (e.g., less than or equal to about 30%, 25%, 20%, 15%, 10%, or 5% (including all values ​​and ranges between them)) after reconstitution, including by any of the methods described herein.

[0127] f.mRNA integrity The reconstituted dried powder formulations described herein, including but not limited to the exemplary embodiments described herein, can provide beneficial effects on mRNA cargo integrity.

[0128] In some embodiments, mRNA retains integrity of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, and 98% or more (including any values ​​and subranges between them) after storage for 1, 2, 3, 4, 5, 6, 7, 8, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 60% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 61% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 62% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 63% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 64% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer.In some embodiments, mRNA maintains 65% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 66% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 67% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 68% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 69% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 70% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer.In some embodiments, mRNA maintains 71% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 72% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 73% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 74% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 75% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 76% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer.In some embodiments, mRNA maintains 77% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 78% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 79% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 80% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 81% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 82% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer.In some embodiments, mRNA maintains 83% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 84% ​​or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 85% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 86% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 87% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 88% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer.In some embodiments, mRNA maintains 89% or more integrity after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer. In some embodiments, mRNA maintains 90% or more integrity for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer.

[0129] In some embodiments, mRNA maintains an integrity of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, and 98% or higher (including any values ​​and partial ranges between them) after being stored at 4°C for at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 months. In some embodiments, mRNA maintains an integrity of 60% or higher after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 61% or higher after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 62% or higher after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 63% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 64% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 65% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 72% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 73% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains 74% or more integrity after being stored at 4°C for at least 6 months.In some embodiments, mRNA maintains 75% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 76% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 78% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 79% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 88% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 89% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 91% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 92% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA retains more than 93% integrity after being stored at 4°C for at least 6 months.In some embodiments, mRNA maintains 94% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 95% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 96% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 97% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 98% or more integrity after being stored at 4°C for at least 6 months. In some embodiments, mRNA maintains 99% or more integrity after being stored at 4°C for at least 6 months.

[0130] In some embodiments, mRNA retains 60% or more integrity, for example 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or more (including any values ​​and subranges between them), after being stored for at least one year at 2–8°C (e.g., 4°C). In some embodiments, mRNA retains 60% or more integrity, for example 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or more (including any values ​​and subranges between them), after being stored for more than one year at 2–8°C (e.g., 4°C).

[0131] In some embodiments, mRNA retains an integrity of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or higher (including any values ​​and partial ranges between them) after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 60% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 61% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 62% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 63% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 64% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains an integrity of 65% or higher after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 72% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 73% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 74% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA retains more than 75% integrity after being stored at 25°C for at least 6 months.In some embodiments, mRNA maintains 76% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 78% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 79% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 88% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 89% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 91% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 92% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 93% or more integrity after being stored at 25°C for at least 6 months.In some embodiments, mRNA maintains 94% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 95% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 96% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 97% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 98% or more integrity after being stored at 25°C for at least 6 months. In some embodiments, mRNA maintains 99% or more integrity after being stored at 25°C for at least 6 months.

[0132] In some embodiments, mRNA maintains an integrity of approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or higher (including any values ​​and partial ranges between them) after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 60% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 61% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 62% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 63% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 64% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains an integrity of 65% or higher after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 66% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 67% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 68% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 69% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 70% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 71% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 72% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 73% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 74% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA retains more than 75% integrity after being stored at -20°C for at least 6 months.In some embodiments, mRNA maintains 76% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 77% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 78% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 79% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 81% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 82% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 83% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 84% ​​or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 85% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 86% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 87% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 88% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 89% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 91% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 92% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 93% or more integrity after being stored at -20°C for at least 6 months.In some embodiments, mRNA maintains 94% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 95% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 96% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 97% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 98% or more integrity after being stored at -20°C for at least 6 months. In some embodiments, mRNA maintains 99% or more integrity after being stored at -20°C for at least 6 months.

[0133] In some embodiments, mRNA retains 60% or more of its integrity after storage at -20°C for at least one year, e.g., 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or more (including any values ​​and subranges between them).

[0134] g. Delivery route In some embodiments, a reconstituted dry powder product containing a sugar or sugar alcohol enables the administration of mRNA-nanoparticles as a liquid formulation in a desired dose. As disclosed herein, the term “desired dose” is a dose that is suitable for the reconstituted dry powder formulation described herein. Pharmaceutical compositions comprising the reconstituted dry powder product described herein may be administered subconjunctivally, sublingually, to a subject requiring it by any one of several routes that effectively deliver an effective amount of the compound. Non-limiting examples of preferred routes of administration include topical, oral, nasal, intrathecal, enteral, oral cavity, sublingual, transdermal, rectal, vaginal, intraocular, and parenteral administration (subcutaneous, intravenous, intramuscular, intrasternal, intracavernosal, intracavitary, and intraurethral injection and / or infusion).

[0135] In some embodiments, the reconstituted dried powder product is suitable for mucosal delivery. In some embodiments, the reconstituted dried powder product is suitable for oral delivery. In some embodiments, the reconstituted dried powder product is suitable for sublingual delivery. In some embodiments, the formulation is suitable for or intranasal delivery. In some embodiments, the reconstituted dried powder product is suitable for oral delivery. In some embodiments, the reconstituted dried powder product is suitable for any delivery route suitable for this disclosure.

[0136] N / P ratio of lipid nanoparticles In some embodiments, the preparation of LNPs involves encapsulating mRNA lipids, which are added to an aqueous buffer containing mRNA at a specific nitrogen (lipid) to phosphate (nucleic acid) ratio (N / P ratio).

[0137] In some embodiments, mRNA and lipids are combined with a pump system that maintains a constant lipid / mRNA(N / P) ratio throughout the process and allows for easy scale-up.

[0138] In some embodiments, one or more LNPs encapsulating mRNA (also called mRNA-loaded LNPs or mRNA-LNPs) have a lipid:mRNA(N / P) ratio in the range of 1–20, 1–15, 1–10, 2–8, 2–6, or 2–4. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1–20. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1–18. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1–16. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1–14. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1–12. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1 to 10. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1 to 8. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 1 to 6. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 20. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 16. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 12. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 8. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 6. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 2 to 4. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 4 to 20.In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 4 to 16. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 4 to 14. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 4 to 12. In some embodiments, one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA(N / P) ratio in the range of 4 to 10. In some embodiments, one or more mRNA-loaded LNPs have a lipid:mRNA(N / P) ratio of 2 or 4. In some embodiments, one or more mRNA-loaded LNPs have a lipid:mRNA(N / P) ratio of 2. In some embodiments, one or more mRNA-loaded LNPs have a lipid:mRNA(N / P) ratio of 4.

[0139] In some embodiments, the lipid nanoparticles encapsulating the mRNA have an N / P ratio of 2 to 6. In some embodiments, the N / P ratio is 3 to 4. In some embodiments, the N / P ratio is 3.

[0140] In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 1. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 2. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 3. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 4. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 5. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 6. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 7. In some embodiments, the dry powder formulation includes lipid nanoparticles added to mRNA in an N:P ratio of approximately 8.

[0141] Encapsulation efficiency The dry powder formulations described herein, including those provided below by further exemplary embodiments, can have unexpectedly improved encapsulation efficiency.

[0142] In some embodiments, one or more mRNA-loaded lipid nanoparticles of the dried powder formulation described herein have an encapsulation efficiency of about 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or more (including any value and partial range between them). In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 70% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 71% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 72% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 73% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 74% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 75% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 76% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 77% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 78% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 79% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 80% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 81% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 82% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 83% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 84% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 85% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 86% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 87% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 88% or more.In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 89% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 90% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 91% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 92% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 93% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 94% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 95% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 96% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 97% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 98% or more. In some embodiments, one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 99% or more.

[0143] In some embodiments, the reconstituted dried powder formulations described herein provide equivalent encapsulation efficiency compared to equivalent non-dried powder formulations. In some embodiments, the encapsulation efficiency of one or more mRNA-loaded lipid nanoparticles in the dried powder formulations described herein does not change significantly during storage of the reconstituted dried powder formulations, including under any exemplary storage conditions, duration, and / or temperature described herein. In some embodiments, the encapsulation efficiency of one or more mRNA-loaded lipid nanoparticles in the reconstituted dried powder formulations does not change beyond about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, and 50% (including all values ​​and ranges between them) (for example, about 30%, 25%, 20%, 15%, 10%, or 5% or less (including all values ​​and ranges between them)) after storage, including under any exemplary storage conditions, duration, and / or temperature described herein.

[0144] In some embodiments of the reconstituted dried powder formulations described herein, equivalent encapsulation efficiency is provided compared to the reconstituted dried powder formulation before reconstitution. In some embodiments, the encapsulation efficiency of one or more mRNA-loaded lipid nanoparticles in the reconstituted dried powder formulations described herein does not change beyond about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40%, 50% (including all values ​​and ranges between them) (for example, about 30%, 25%, 20%, 15%, 10%, or 5% or less (including all values ​​and ranges between them)) after reconstitution, including by any of the methods described herein.

[0145] LNP size and size adjustment before spray drying As described herein, the dried powder formulations described herein can be formulated to provide lipid nanoparticle sizes optimal for a variety of applications, including those provided by further exemplary embodiments below.

[0146] Suitable mRNA-loaded lipid nanoparticles can be produced in various sizes.

[0147] In some embodiments, the size of mRNA-loaded lipid nanoparticles before spray drying is determined by the length of the maximum diameter of the lipid nanoparticles. In some embodiments, the mRNA-loaded lipid nanoparticles have a pre-spray-dried size of about 250 nm or less (e.g., about 250 nm, about 225 nm, about 200 nm, about 175 nm, about 150 nm, about 125 nm, about 100 nm, about 90 nm, about 80 nm, about 75 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 25 nm, about 20 nm, or about 10 nm or less (including all values ​​and ranges in between)). In some embodiments, preferred liposomes have a size in the range of about 10 nm to about 250 nm (e.g., in the range of about 10 nm to about 225 nm, about 10 nm to about 200 nm, about 10 nm to about 175 nm, about 10 nm to about 150 nm, about 10 nm to about 125 nm, about 10 nm to about 100 nm, about 10 nm to about 75 nm, or about 10 nm to about 50 nm). In some embodiments, mRNA-loaded lipid nanoparticles have a pre-spray dried size in the range of about 100 nm to about 250 nm (e.g., in the range of about 100 nm to about 225 nm, about 100 nm to about 200 nm, about 100 nm to about 175 nm, or about 100 nm to about 150 nm). In some embodiments, the mRNA-loaded lipid nanoparticles have a pre-spray dry size in the range of about 10 nm to about 100 nm (e.g., in the range of about 10 nm to about 90 nm, about 10 nm to about 80 nm, about 10 nm to about 70 nm, about 10 nm to about 60 nm, or about 10 nm to about 50 nm). In certain embodiments, the mRNA-loaded lipid nanoparticles have a pre-spray dry size of less than about 100 nm.

[0148] Various alternative methods known in the art are available for resizing liposome populations. One such resizing method is described in U.S. Patent No. 4,737,323, incorporated herein by reference. Sonication of liposome suspensions by either in-bath or probe sonication results in a gradual reduction in size to small ULVs with a diameter of less than approximately 0.05 microns. Homogenization is another method that relies on shear energy to fragment larger liposomes into smaller ones. In a typical homogenization procedure, the MLVs are recirculated through a standard emulsion homogenizer until a selected liposome size of approximately 0.1–0.5 microns is observed. Liposome size can be determined by quasi-electrical light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles can be performed alternately with QELS evaluation to facilitate efficient liposome synthesis.

[0149] Spray drying process Spray drying is a commonly used, economical, and well-established technique for producing dried powder products for various modalities such as low molecular weight, peptides, and proteins. This technique is continuous, scalable, suitable for heat-sensitive materials, can produce consistent dried powder products, and can be automated. Various sugars, such as lactose and mannitol, are commonly used as carrier excipients to facilitate the spray drying process. For example, in the compositions and processes described herein, the beneficial properties of mannitol as an excipient for spray drying mRNA formulations include (i) an effect on the viscoelastic properties related to sputum, (ii) an increase in water content driven by an osmotic gradient, (iii) lower hygroscopicity compared to some other sugars such as lactose, and (iv) a non-reducing sugar that does not contain an aldehyde group. However, amino acids such as leucine, isoleucine, and trileucine are used to improve the dispersibility of dried powder products and reduce MMAD. Spray drying is continuous and scalable, suitable for heat-sensitive materials, and can produce consistent DPP; however, there are few reports on excipient screening and optimization of the formulation properties and aerosol performance of spray-dried mRNA LNPs.

[0150] Various spray drying processes can be used to implement this disclosure.

[0151] This process generally involves removing water from a composition by passing it through a device in liquid form. For example, a liquid formulation containing the composition of interest passes through a narrow inlet atomizer nozzle into a first chamber, which is a drying chamber. Typically, the liquid formulation passes through in a steady flow. The liquid formulation is sprayed into the drying chamber as small droplets. A flow of heated air or gas is also introduced into the drying chamber, forming an airflow. The passage of the formulation by this heated flow disperses the incoming droplets and dries them into solid particle form. This product is then led to a second chamber by a flow through a connector or pipe. The second chamber is a cyclone powder collector. Here, air circulation generates a cyclone, and the powder particles are collected in a collection container attached to the outlet end via a vortex. The cyclone chamber is attached to an exhaust fan that helps cool the components. The inlet and outlet temperatures are adjustable by the operator. The respective inlet and outlet temperatures, chamber temperature, liquid supply flow rate (aspirator %), pressure, heating airflow properties, and most importantly, the composition of the liquid feed are suitably adjusted for optimal drying of any particulate matter.

[0152] In some embodiments, the inlet temperature is adjustable within the range of 40°C to 200°C. In some embodiments, the outlet temperature is in the range of 20°C to 70°C. The relative pressure between the pump and the aspirator is also adjustable by the operator. In some embodiments, the inlet temperature was adjusted to 55°C. In some embodiments, the inlet temperature was adjusted to 60°C. In some embodiments, the inlet temperature was adjusted to 61°C. In some embodiments, the inlet temperature was adjusted to 62°C. In some embodiments, the inlet temperature was adjusted to 63°C. In some embodiments, the inlet temperature was adjusted to 64°C. In some embodiments, the inlet temperature was adjusted to 65°C. In some embodiments, the inlet temperature was adjusted to 66°C. In some embodiments, the inlet temperature was adjusted to 67°C. In some embodiments, the inlet temperature was adjusted to 68°C. In some embodiments, the inlet temperature was adjusted to 69°C. In some embodiments, the inlet temperature was adjusted to 70°C.

[0153] In some embodiments, the inlet temperature is adjusted to 70°C to 200°C for spray-drying mRNA-lipid nanoparticles. In some embodiments, the inlet temperature is adjusted to 80°C to 200°C. In some embodiments, the inlet temperature is adjusted to 90°C to 200°C. In some embodiments, the inlet temperature is adjusted to 95°C to 180°C. In some embodiments, the inlet temperature is adjusted to 95°C to 160°C. In some embodiments, the inlet temperature is adjusted to 90°C to 150°C. In some embodiments, the inlet temperature is adjusted to 90°C to 120°C. In some embodiments, the inlet temperature is adjusted to 90°C to 100°C. In some embodiments, the inlet temperature is 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C (including any value and partial ranges between them).

[0154] In some embodiments, the outlet temperature is in the range of 20°C to 70°C. In some embodiments, the outlet temperature is 30°C to 60°C. In some embodiments, the outlet temperature is 20°C to 50°C. In some embodiments, the outlet temperature is 30°C to 50°C. In some embodiments, the outlet temperature is 40°C to 50°C. In some embodiments, the outlet temperature is 45°C to 50°C.

[0155] Spray drying can be carried out using any suitable spray drying apparatus. As is known to those skilled in the art, various spray drying apparatuses are commercially available and can be used to carry out this disclosure. Examples of commercially available equipment suitable for this disclosure include, but are not limited to, the following: Mini Spray Dryer B-290; Nano Spray Dryer B-90 (Buchi); Anhydro MicraSpray Dryer GMP; Anhydro MicraSpray Dryer Aseptic series (SPX FLOW); MDL-50 and MDL-015 (Fujisaki Electric); Versatile Mini Sprayer Dryer GAS410 (Yamato Scientific America); LSD-1500 Mini spray dryer, MSD-8 Multi-functional laboratory spray dryer; PSD-12 Precision pharmacy spray dryer (Changzhou Xiandao Drying Equipment Co. Ltd); TALL FORM DRYER®; Multi-Stage Dryer; COMPACT DRYER®; FILTERMAT® Spray Dryer; VERSATILE-SD®; Fluidized Spray Dryer; MOBILE MINOR®; SDMICRO®; PRODUCTION MINOR® (trademark) (manufactured by GEA Process Engineering) and many other products. Convenient scale-ups from laboratory to industrial production scales are also available from some of these manufacturers.

[0156] dry powder The dried powder prepared according to this disclosure contains multiple spray-dried particles. Residual moisture content, aerosol performance, and physiological and chemical stability are important parameters for spray-dried pharmaceuticals. These are determined by the weight loss of the sample after heating and drying, using the following formula:

number

[0157] Generally, an acceptable range for particle size distribution is maintained for the uniformity of the administration of the active pharmaceutical ingredient of the formulation. In some embodiments, the particles of a dry powder formulation suitable for pulmonary administration contain trehalose. In some embodiments, the particles of a dry powder formulation suitable for pulmonary administration containing trehalose are 5 micrometers or less. In some embodiments, spraying lipid nanoparticles with an aqueous alcohol solution helps to reduce the particle size of DPPs. In some embodiments, spraying lipid nanoparticles with an aqueous alcohol solution containing a sugar or sugar alcohol helps to reduce the particles of DPPs. In some embodiments, the sugar is mannitol, xylitol, lactose, sucrose, or trehalose. In some embodiments, the sugar is trehalose. In some embodiments, the sugar or sugar alcohol is mannitol. In some embodiments, the sugar is sucrose. In some embodiments, the sugar or sugar alcohol is xylitol. In some embodiments, the sugar is lactose.

[0158] In some embodiments, the particle size (e.g., average particle size) of the dry powder is 0.5 to 10 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder formulation is 1 to 8 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder formulation is 1 to 7 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder formulation is 1 to 6 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is 1 to 5 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is 1 to 4 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is 1 to 3 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is 1 to 2 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is approximately 1 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is approximately 2 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is approximately 3 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 4 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 5 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 6 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 7 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 8 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 9 μm. In some embodiments, the particle size (e.g., average particle size) of the dry powder is about 10 μm.

[0159] The primary particle size (e.g., average particle size) distribution of spray-dried particles is measured by dynamic light scattering and expressed as the Z-mean. The Z-mean, also known as the cumulant size, is calculated from the intensity-weighted distribution of particle size and is given by the following equation:

number

[0160] On the other hand, the polydispersity index (PDI) is a measure of the molecular weight distribution of a given particulate sample. In some embodiments, the polydispersity index of glycerol and propylene glycol-based LNPs is less than 0.2. In some embodiments, the polydispersity index of glycerol and propylene glycol-based LNPs is about 0.1. In some embodiments, the polydispersity index of glycerol and propylene glycol-based LNPs is less than about 0.1.

[0161] mRNA In some embodiments, mRNA constitutes more than about 2% by weight of the dry powder formulation. In some embodiments, mRNA constitutes more than about 3% by weight of the dry powder formulation. In some embodiments, mRNA constitutes more than about 4% by weight of the dry powder formulation.

[0162] The dried powder formulations of mRNA according to this disclosure have been observed to have high stability. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 80%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 81%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 82%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 83%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 84%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 85%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 86%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 87%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 88%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 89%. In some embodiments, the mRNA in the dried powder formulation maintains an integrity of over 90%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 91%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 92%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 93%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 94%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 95%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 96%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 97%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 98%. In some embodiments, the mRNA in the dry powder formulation maintains an integrity of over approximately 99%.

[0163] mRNA integrity is desirable to be maintained after multiple freeze-thaw cycles and / or over a long period of time to maintain therapeutic benefits. In some embodiments, mRNA maintains 80% or more integrity after being stored at room temperature for more than 6 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at room temperature for more than 3 months. In some embodiments, mRNA maintains 80% or more integrity after being stored at room temperature for more than 1 year. In some embodiments, mRNA maintains 90% or more integrity after being stored at room temperature for more than 6 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at room temperature for more than 3 months. In some embodiments, mRNA maintains 90% or more integrity after being stored at room temperature for more than 1 year.

[0164] In some embodiments, mRNA maintains an integrity of 80%, 85%, 90%, 92%, 94%, 96%, 98% or higher (including any values ​​and subranges between them) after storage at 2–8°C (e.g., 4°C) for 4 weeks or more, e.g., 1 month, 3 months, 6 months, 9 months, or 1 year or more (including any values ​​and subranges between them). In some embodiments, mRNA maintains an integrity of 80% or higher after storage at 2–8°C (e.g., 4°C) for 4 weeks or more. In some embodiments, mRNA maintains an integrity of 80% or higher after storage at 2–8°C (e.g., 4°C) for 3 months or more. In some embodiments, mRNA maintains an integrity of 80% or higher after storage at 2–8°C (e.g., 4°C) for 6 months or more. In some embodiments, mRNA maintains an integrity of 80% or higher after storage at 2–8°C (e.g., 4°C) for 1 year or more. In some embodiments, mRNA maintains 85% or more integrity after being stored at 2-8°C (e.g., 4°C) for 4 weeks or more. In some embodiments, mRNA maintains 85% or more integrity after being stored at 2-8°C (e.g., 4°C) for 3 months or more. In some embodiments, mRNA maintains 85% or more integrity after being stored at 2-8°C (e.g., 4°C) for 6 months or more. In some embodiments, mRNA maintains 85% or more integrity after being stored at 2-8°C (e.g., 4°C) for 1 year or more. In some embodiments, mRNA maintains 90% or more integrity after being stored at 2-8°C (e.g., 4°C) for 4 weeks or more. In some embodiments, mRNA maintains 90% or more integrity after being stored at 2-8°C (e.g., 4°C) for 3 months or more. In some embodiments, mRNA maintains 90% or more integrity after being stored at 2-8°C (e.g., 4°C) for 6 months or more. In some embodiments, mRNA maintains over 90% integrity after being stored at 2-8°C (e.g., 4°C) for more than one year. In some embodiments, mRNA maintains over 95% integrity after being stored at 2-8°C (e.g., 4°C) for more than four weeks. In some embodiments, mRNA maintains over 95% integrity after being stored at 2-8°C (e.g., 4°C) for more than three months.In some embodiments, mRNA maintains over 95% integrity after being stored at 2–8°C (e.g., 4°C) for more than 6 months. In some embodiments, mRNA maintains over 95% integrity after being stored at 2–8°C (e.g., 4°C) for more than 1 year.

[0165] In some embodiments, mRNA retains more than 80% integrity after being stored at -80°C following three freeze-thaw cycles. In some embodiments, mRNA retains more than 90% integrity after being stored at -80°C following three freeze-thaw cycles.

[0166] As used herein, the phrase "mRNA maintains at least x% integrity after storage" means that mRNA integrity does not decrease by more than (100-x)% after storage.

[0167] mRNA synthesis The mRNA according to this disclosure may be synthesized according to any of a variety of known methods. Various methods are described in the published U.S. Patent Application Publication No. 2018 / 0258423, all of which are incorporated herein by reference, and may be used to practice the invention. For example, the mRNA according to this disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed on a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0168] In some embodiments, the preferred mRNA sequence is an mRNA sequence encoding a protein or peptide. In some embodiments, the preferred mRNA sequence is codon-optimized for efficient expression in human cells. In some embodiments, the preferred mRNA sequence is a naturally occurring sequence or a wild-type sequence. In some embodiments, the preferred mRNA sequence encodes a protein or peptide containing one or more mutations in its amino acid sequence. Exemplary mRNA coding sequences and their corresponding amino acid sequences are shown below.

[0169] Example of mRNA construct design X-mRNA coding region -Y 5' and 3' UTR sequences X(5'UTR array) = [ka] Y(3'UTR array) = [ka]

[0170] This disclosure can be used to deliver mRNA of various lengths. In some embodiments, this disclosure can be used to deliver in vitro synthesized mRNA of lengths of about 0.5kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb, 20kb, 30kb, 40kb, or 50kb or greater (including any values ​​and subranges between them). In some embodiments, the disclosure may be used to deliver in vitro synthesized mRNA in the range of lengths of about 1–20kb, about 1–15kb, about 1–10kb, about 5–20kb, about 5–15kb, about 5–12kb, about 5–10kb, about 8–20kb, or about 8–50kb.

[0171] In some embodiments, the DNA template is transcribed in vitro for mRNA preparation according to this disclosure. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired mRNA and a desired nucleotide sequence for terminal signaling.

[0172] nucleotide Various naturally occurring or modified nucleotides may be used to generate mRNA according to this disclosure. In some embodiments, mRNA may be naturally occurring nucleosides (or unmodified nucleotides; e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-dea Zaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite bonds) or comprising them.

[0173] In some embodiments, preferred mRNA may contain skeletal modifications, sugar modifications, and / or base modifications. For example, modified nucleotides include modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl Ru-adenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl Examples of ur-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, queosin, beta-D-mannosyl-queosin, weybutoxosin, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine may be, but are not limited to, these.The preparation of such analogues is known to those skilled in the art from, for example, U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, U.S. Patent Nos. 5,262,530 and 5,700,642, and these disclosures are incorporated by reference as a whole.

[0174] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. These non-standard nucleotide residues may include, for example, 5-methylcytidine ("5mC"), pseudouridine ("yU"), and / or 2-thiouridine ("2sU"). For a discussion of such residues and their incorporation into mRNA, see, for example, U.S. Patent No. 8,278,036 or International Publication No. 2011 / 012316. The mRNA may be defined as RNA in which 25% of the U residues are 2-thiouridine and 25% of the C residues are 5-methylcytidine. Teachings relating to the use of RNA are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and International Publication No. 2011 / 012316, both of which are incorporated herein by reference as a whole. The presence of non-standard nucleotide residues can make mRNA more stable and / or less immunogenic than control mRNA having the same sequence but containing only standard residues. In further embodiments, mRNA may contain one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurinecytosine, and combinations of these modifications and other nucleic acid base modifications. Some embodiments may further include additional modifications to the furanose ring or nucleic acid bases. Additional modifications may include, for example, sugar modifications or substitutions (e.g., 2'-O-alkyl modifications, one or more locked nucleic acids (LNAs)). In some embodiments, RNA may form complexes or hybridize with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications may include, but are not limited to, 2'-deoxy-2'-fluoro modification, 2'-O-methyl modification, 2'-O-methoxyethyl modification, and 2'-deoxy modification.In some embodiments, any of these modifications may be present in 0 to 100% of the nucleotide, for example, individually or in combination, in amounts greater than 0%, greater than 1%, greater than 10%, greater than 25%, greater than 50%, greater than 75%, greater than 85%, greater than 90%, greater than 95%, or greater than 100% of the component nucleotide (including any values ​​and subranges between them).

[0175] In some embodiments, mRNA may contain RNA backbone modifications. Typically, backbone modifications are chemical modifications of the phosphate groups in the nucleotide backbone contained in RNA. Exemplary backbone modifications include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphates, and positively charged guanidium groups. This means replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0176] In some embodiments, mRNA may contain sugar modifications. Typical sugar modifications are chemical modifications of sugars in nucleotides, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, and 2'-deoxy-2'- The product contains, but is not limited to, sugar modifications selected from the group consisting of C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracithidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azido tripphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0177] Post-synthesis processing Typically, a 5' cap and / or 3' tail may be added after synthesis. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect mRNA from exonuclease degradation.

[0178] The 5' cap is typically added as follows: first, a phosphatase at the end of the RNA removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, creating a 5'5'5 triphosphate bond; and then, the 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Further cap structures are described in U.S. Patent Application Publication 2016 / 0032356 and U.S. Patent Application Publication 2018 / 0125989, which are incorporated herein by reference.

[0179] Typically, the tail structure includes a poly(A) and / or poly(C) tail. The poly-A or poly-C tail on the 3' end of mRNA typically contains at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, and at least 550 a The compound comprises adenosine or cytosine nucleotide, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotide (including any values ​​and subranges between them).In some embodiments, the poly-A or poly-C tail contains approximately 10 to 800 adenosine or cytosine nucleotides (for example, approximately 10 to 200 adenosine or cytosine nucleotides, approximately 10 to 300 adenosine or cytosine nucleotides, approximately 10 to 400 adenosine or cytosine nucleotides, approximately 10 to 500 adenosine or cytosine nucleotides, approximately 10 to 550 adenosine or cytosine nucleotides, approximately 10 to 600 adenosine or cytosine nucleotides, approximately 50 to 600 adenosine or cytosine nucleotides, approximately 100 to 600 adenosine or cytosine nucleotides, approximately 150 to 600 adenosine or cytosine nucleotides, approximately 20 The adenosine or cytosine nucleotides may be 0 to 600, about 250 to 600, about 300 to 600, about 350 to 600, about 400 to 600, about 450 to 600, about 500 to 600, about 10 to 150, about 10 to 100, about 20 to 70, or about 20 to 60. In some embodiments, the tail structure includes combinations of poly(A) and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (including any values ​​and subranges between them) of adenosine nucleotides. In some embodiments, the tail structure contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (including any values ​​and subranges between them) of cytosine nucleotides.

[0180] As described herein, the addition of a 5' cap and / or 3' tail facilitates the detection of interrupted mRNA transcripts generated during in vitro synthesis, because without the addition of the cap and / or tail, the size of the interrupted mRNA transcripts might be too small to detect. Therefore, in some embodiments, the 5' cap and / or 3' tail are added to mRNA synthesized before the mRNA is tested for purity (e.g., the level of interrupted transcripts present in the mRNA). In some embodiments, the 5' cap and / or 3' tail are added to mRNA synthesized before the mRNA is purified as described herein. In other embodiments, the 5' cap and / or 3' tail are added to mRNA synthesized after the mRNA has been purified as described herein.

[0181] mRNA synthesized according to this disclosure may be used without further purification. In particular, mRNA synthesized according to this disclosure may be used without the step of removing the shorter. In some embodiments, mRNA synthesized according to this disclosure may be further purified. Various methods may be used to purify mRNA synthesized according to this disclosure. For example, the purification of mRNA may be carried out by centrifugation, filtration and / or chromatography. In some embodiments, the synthesized mRNA is purified by ethanol precipitation, or filtration, or chromatography, or gel purification or any other preferred means. In some embodiments, mRNA is purified by HPLC. In some embodiments, mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution well known to those skilled in the art. In some embodiments, mRNA is purified using tangential flow filtration. Suitable purification methods include those described in U.S. Patent Application Publication No. 2016 / 0040154, U.S. Patent Application Publication No. 2015 / 0376220, U.S. Patent Application Publication No. 2018 / 0251755, U.S. Patent Application Publication No. 2018 / 0251754, U.S. Provisional Patent Application No. 62 / 757,612 filed November 8, 2018, and U.S. Provisional Patent Application No. 62 / 891,781 filed August 26, 2019.

[0182] In some embodiments, mRNA is purified before capping and tailing. In some embodiments, mRNA is purified after capping and tailing. In some embodiments, mRNA is purified both before and after capping and tailing.

[0183] In some embodiments, mRNA is purified by centrifugation either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by filtration either either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by tangential flow filtration (TFF) either either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by chromatography either either before or after capping and tailing, or both before and after.

[0184] Additional lipids In some embodiments, the lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the lipid nanoparticles further comprise one or more cholesterol-based lipids. In some embodiments, one or more cationic lipids constitute about 30-70% of the total lipids in the LNP in mol%. In some embodiments, one or more PEG-modified lipids constitute about 1-15% of the total lipids in the LNP in mol%. In some embodiments, one or more non-cationic lipids constitute about 10-40% of the total lipids in the LNP in mol%. In some embodiments, one or more cholesterol-based lipids constitute about 5-40% of the total lipids in the LNP in mol%.

[0185] In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids in lipid nanoparticles is approximately 60:25:10:5. In some embodiments, the molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids in lipid nanoparticles is approximately 40:25:30:5.

[0186] Exemplary lipids are described herein.

[0187] Cationic lipids As used herein, the term "cationic lipid" refers to any of several lipid species having a net positive charge at a selected pH, such as physiological pH.

[0188] Suitable cationic lipids for use in the compositions and methods of the present disclosure include cationic lipids as described in WO 2010 / 144740 pamphlet, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present disclosure have the following compound structure:

Chem.

[0189] Other suitable cationic lipids for use in the compositions and methods of the present disclosure include ionizable cationic lipids as described in WO 2013 / 149140 pamphlet, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present disclosure have one of the following formulas:

Chem.

[0190] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids described as amino alcohol lipidoids in International Publication No. 2010 / 053572, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure utilize cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts.

[0191] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2016 / 118725, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts.

[0192] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2016 / 118724, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts.

[0193] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids having the formula 14,25-ditridecyl15,18,21,24-tetraaza-octatriacontane and pharmaceutically acceptable salts thereof.

[0194] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2013 / 063468 and International Publication No. 2016 / 205691, respectively, which are incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt (wherein R L Each example is independently and optionally substituted C6~C 40 It includes an alkenyl. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts.

[0195] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] or its pharmaceutically acceptable salt (wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each R Ais independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclic, optionally substituted 3-14 member heterocyclic, optionally substituted C6-14 aryl, optionally substituted 5-14 member heteroaryl or halogen; and each R B is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclic, optionally substituted 3-14 member heterocyclic, optionally substituted C6-14 aryl, optionally substituted 5-14 member heteroaryl or halogen). In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the following chemical formula structure, "Target 23": [Chemical formula] (Target 23) and pharmaceutically acceptable salts thereof.

[0196] Other suitable cationic lipids for use in the compositions and methods of the present disclosure include cationic lipids as described in WO 2016 / 004202 pamphlet, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the following compound structure: [Chemical formula] or pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the following compound structure: [Chemical formula] or pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the following compound structure: [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0197] Other suitable cationic lipids for use in the compositions and methods of the present disclosure include cationic lipids as described in U.S. Provisional Patent Application No. 62 / 758,179, which is hereby incorporated by reference herein. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid of the following formula: [Chemical formula] or a pharmaceutically acceptable salt thereof (wherein R 1 and R 2 are each independently H or C1-C6 aliphatic; each m is an integer having a value of 1-4; each A is independently a covalent bond or an arylene; each L 1 is independently an ester, thioester, disulfide or anhydride group; each L 2 is independently C2-C 10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C6-C 20 aliphatic). In some embodiments, the compositions and methods of the present disclosure include a cationic lipid of the following formula: [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid of the following formula: [Chemical formula] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid of the following formula: [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0198] Other suitable cationic lipids for use in the compositions and methods of this disclosure include those described by reference herein, J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277. In some embodiments, the cationic lipids of the compositions and methods of this disclosure are cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0199] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the following compound structure:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0200] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structures: [ka] and its pharmaceutically acceptable salts.

[0201] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] or its pharmaceutically acceptable salt (wherein L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NRa -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O- and L 1 or L 2 Other examples include -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or direct bond; G 1 and G 2 Each of these is independently of the non-substituted C1~C 12 Alkylene and C1~C 12 It is an alkenylene; G 3 C1~C 24 Alkylene, C1~C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a H or C1~C 12 It is alkyl; R 1 and R 2 Each of these is independent of C6~C 24 Alkyl or C6-C 24 It is an alkenil; R 3 H, OR 5 , CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 And; R 4 C1~C 12 It is alkyl; R 5 (where x is H or C1-C6 alkyl; and x is 0, 1, or 2).

[0202] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2017 / 117528, which is incorporated herein by reference.

[0203] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0204] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0205] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0206] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2017 / 049245, which is incorporated herein by reference.

[0207] In some embodiments, the cationic lipid of the compositions and methods of the present disclosure is one compound of the following formulas: [ka] and its pharmaceutically acceptable salts.

[0208] For any one of these four equations, R 4It is independently, -(CH2) n Q and -(CH2) n Selected from CHQR; Q is -OR, -OH, -O(CH2) n Selected from the group consisting of N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and the hetero rings; and n is 1, 2, or 3.

[0209] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0210] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0211] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0212] In certain specific embodiments, the compositions and methods of the present disclosure are cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0213] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cationic lipids as described in International Publication No. 2017 / 173054 and International Publication No. 2015 / 095340, which are respectively incorporated herein by reference.

[0214] In certain specific embodiments, the compositions and methods of the present disclosure are cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0215] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0216] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0217] In some embodiments, compositions and methods of the present disclosure include cationic lipids having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0218] Other suitable cationic lipids for use in the compositions and methods of this disclosure include cleavable cationic lipids as described in International Publication No. 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] (In the formula, R1 is selected from the group consisting of imidazole, guanidium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; R2 is one of the following two formulas: [ka] Selected from the group consisting of one of the following; and R3 and R4 are each independently substituted by arbitrary choice with indeterminate saturated or unsaturated C6~C 20 Alkyl and optionally substituted, unspecified saturated or unsaturated C6-C 20 Selected from the group consisting of acyls; and n includes 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater).

[0219] In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structure, "HGT4001": [ka] and its pharmaceutically acceptable salts.

[0220] In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structure, "HGT4002": [ka] and its pharmaceutically acceptable salts.

[0221] In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structure, "HGT4003": [ka] and its pharmaceutically acceptable salts.

[0222] In some embodiments, the compositions and methods of the present disclosure include cationic lipids having the following compound structure, "HGT4004": [ka] and its pharmaceutically acceptable salts.

[0223] In some embodiments, the compositions and methods of the present disclosure include a cationic lipid "HGT4005" having the following compound structure: [ka] and its pharmaceutically acceptable salts.

[0224] Other suitable cationic lipids for use in the compositions and methods of this disclosure include HEPES-based disulfide cationic lipids having a piperazine core, as described in International Publication No. 2022 / 221688, which is incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] and its pharmaceutically acceptable salts. In some embodiments, the compositions and methods of the present disclosure include cationic lipids of the following formula: [ka] and its pharmaceutically acceptable salts. In some embodiments, the compositions and methods of the present disclosure include cationic lipids of the following formula: [ka] and its pharmaceutically acceptable salts.

[0225] Other suitable cationic lipids for use in the compositions and methods of this disclosure include the cationic lipids described by reference in Dong et al., PNAS, 2014, 111(11):3955-3960 and U.S. Patent No. 9,512,073. In some embodiments, the compositions and methods of this disclosure use cationic lipids of the following formula: [ka] and its pharmaceutically acceptable salts. In some embodiments, the compositions and methods of the present disclosure include cationic lipids of the following formula: [ka] and its pharmaceutically acceptable salts.

[0226] Other suitable cationic lipids for use in the compositions and methods of this disclosure include the cleavable cationic lipids described in U.S. Provisional Patent Application No. 62 / 672,194, filed on 16 May 2018 and incorporated herein by reference. In some embodiments, the compositions and methods of this disclosure include cationic lipids having any of the general formulas or structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in U.S. Provisional Patent Application No. 62 / 672,194.

[0227] In some embodiments, compositions and methods of the present disclosure include cationic lipids having a structure according to formula (I'), [ka] (In the formula, R X These are independently -H and -L 1 -R 1 or -L 5A -L 5B -B' is; L 1 、L 2 、and L 3 each independently is a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or -C(O)NR L -; each L 4A and L 5A each independently is -C(O)-, -C(O)O-, or -C(O)NR L -; each L 4B and L 5B each independently is C1-C 20 alkylene; C2-C 20 alkenylene; or C2-C 20 alkynylene; each B and B' is NR 4 R 5 or a 5- to 10-member nitrogen-containing heteroaryl; each R 1 、R 2 、and R 3 each independently is C6-C 30 alkyl, C6-C 30 alkenyl, or C6-C 30 alkynyl; each R 4 and R 5 each independently is hydrogen, C1-C 10 alkyl; C2-C 10 alkenyl; or C2-C 10 alkynyl; and each R L each independently is hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, or C2-C 20 alkynyl).

[0228] In some embodiments, the compositions and methods of the present disclosure are the cationic lipid of compound (139) of U.S. Patent Application No. 62 / 672,194 having the following compound structure:

Chemical formula

[0229] In some embodiments, the compositions and methods of the present disclosure include a cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). (Feigner et al. (Proc. Nat'l Acad. Sci. 84,7413 (1987); U.S. Patent No. 4,897,355) incorporated herein by reference). Other cationic lipids suitable for the compositions and methods of this disclosure include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-l-propaneaminium ("DOSPA") (Behr et al. Proc. Nat'l Acad. Sci. 86,6982 (1989), U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0230] Additional exemplary cationic lipids suitable for the compositions and methods of the present disclosure include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium Muchloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyroxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholesta-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholesta-5-en-3-beta- Oxy)-3'-oxapentoxy)-3-dimethyl(cis,cis-9',1-2'-octadecadienoxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleyloxy-N,N-dimethylpropylamine ("DLinDAP"); l,2-N,N'-dilinoleylcarbamyl-3-dimethyl Aminopropane ("DLincarbDAP"); l,2-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-Dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-Cholesta-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12dien-1-yloxy]propane-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholesta-5-en-3-yloxy]octyl l)oxy)-N,N-dimethyl l-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholesta-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl 3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane -1-amine ("Octyl-CLinDMA(2S)"); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,l2-dien-1-yl)-l,3-dioxolane-4-yl)-N,N-dimethylethaneamine ("DLin-KC2-DMA") (as incorporated herein by reference in International Publication No. 2010 / 042877; Semple See also et al., Nature Biotech. 28:172-176 (2010) (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); see International Publication No. 2005 / 121348). In some embodiments, one or more cationic lipids include at least one imidazole, dialkylamino, or guanidinium moiety.

[0231] In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present disclosure include 2,2-dilinoleyl-1-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100"); and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0232] In some embodiments, the compositions and methods of the present disclosure are cationic lipids known as ALC-0315 ([(4-hydroxybutyl)azanejyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)), which are synthetic lipids having the following chemical structure: [ka] and its pharmaceutically acceptable salts.

[0233] In some embodiments, the compositions of the present disclosure comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% (including any values ​​and partial ranges between them) of the total lipid content in the composition, for example, as measured by the weight of lipid nanoparticles. In some embodiments, the compositions of the present disclosure comprise one or more cationic lipids that constitute about 30–70% of the total lipid content in the composition, for example, as measured by the weight of lipid nanoparticles (e.g., about 30–65%, about 30–60%, about 30–55%, about 30–50%, about 30–45%, about 30–40%, about 35–50%, about 35–45%, or about 35–40% (including any value and partial ranges between them)). In some embodiments, the compositions of the present disclosure comprise one or more cationic lipids that constitute about 30–70% of the total lipid content in the composition, for example, as measured by mol% of lipid nanoparticles (e.g., about 30–65%, about 30–60%, about 30–55%, about 30–50%, about 30–45%, about 30–40%, about 35–50%, about 35–45%, or about 35–40% (including any value and partial ranges between them)).

[0234] Noncationic / Helper Lipids In some embodiments, the provided liposomes contain one or more noncationic ("helper") lipids. As used herein, the term "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the term "anionic lipid" refers to any of several lipid species having a net negative charge at selected H, such as physiological pH. Examples of noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleymi Examples include, but are not limited to, domethyl-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof.

[0235] In some embodiments, the noncationic lipid is DPPC. In some embodiments, the noncationic lipid is DOPE. In some embodiments, the noncationic lipid is DEPE.

[0236] In some embodiments, such noncationic lipids may be used alone, but are preferably used in combination with other lipids, such as cationic lipids. In some embodiments, noncationic lipids may constitute about 5% to about 90%, or about 10% to about 70%, of the total lipids present in the liposomes in molar ratio. In some embodiments, noncationic lipids are neutral lipids, i.e., lipids that have no net charge under the conditions in which the composition is formulated and / or administered. In some embodiments, the percentage of noncationic lipids in the liposomes may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0237] Cholesterol-based lipids In some embodiments, the provided liposomes contain one or more cholesterol-based lipids. For example, preferred cholesterol-based lipids include cholesterol, such as DC-Choi(N,N-dimethyl-N-ethylcarboxamide cholesterol), l,4-bis(3-N-oleylaminopropyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179,280 (1991); Wolf et al. BioTechniques 23,139 (1997); U.S. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipids may constitute about 2% to about 30% or about 5% to about 20% of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

[0238] PEG modified lipid The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), either alone or preferably in combination with other lipid formulations containing a transport medium (e.g., lipid nanoparticles), is also intended by this disclosure. The intended PEG-modified lipids include C6-C 20 The addition of such components may prevent complex aggregation and may also provide a means to increase circulating lifetime and enhance delivery of the lipid-nucleic acid composition to target tissue (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly replaced from the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful replaceable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of this disclosure may comprise molar ratios of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in the liposome transport medium. In some embodiments, one or more PEG-modified lipids account for approximately 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids account for approximately 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids account for approximately 6% of the total lipids by molar ratio.

[0239] polymer In some embodiments, the preferred delivery medium is formulated using a polymer as a carrier, either alone or in combination with other carriers containing various lipids described herein. Thus, in some embodiments, the liposome delivery medium also includes polymer-containing nanoparticles, as used herein. Suitable polymers include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, protamines, pegylated protamines, PLLs, pegylated PLLs, and polyethyleneimines (PEIs). When a PEI is present, it may be a branched PEI with a molecular weight in the range of 10 to 40 kDa, for example, a 25 kDa branched PEI (Sigma #408727).

[0240] Exemplary combinations of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids are described in the Examples section. For example, preferred lipid solutions include CKK-E10, DOPE, cholesterol, and DMG-PEG2K; CKK-E12, DOPE, cholesterol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, cholesterol, and DMG-PEG2K; HGT5001, DOPE, cholesterol, and DMG-PEG2K; OF-02, DOPE, cholesterol, and DMG-PEG2K; GL-HEPES-E3-E12-DS-4-E10, DOPE, cholesterol, and DMG-P EG2K;CKK-E10, DPPC, cholesterol, and DMG-PEG2K;CKK-E12, DPPC, cholesterol, and DMG-PEG2K;C12-200, DPPC, cholesterol, and DMG-PEG2K;HGT5000, DPPC, cholesterol, and DMG-PEG2K;HGT5001, DPPC, cholesterol, and DMG-PEG2K;OF-02, DPPC, cholesterol, and DMG-PEG2K;or GL-HEPES-E3-E12-DS-4-E10, cholesterol, and DMG-PEG2K may be included. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including the lipid mixture, and the relative molar ratios of such lipids to each other are based on the characteristics of the selected lipids and the properties and characteristics of the encapsulated mRNA. Further considerations include, for example, alkyl chain saturation and the size, charge, pH, pKa, membrane fusion activity, and toxicity of the selected lipids. Therefore, the molar ratios can be adjusted as appropriate.

[0241] In some embodiments, LNPs are produced in a specific molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids. In some embodiments, the lipid nanoparticles have a molar ratio of 60:25:10:5 for cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids. In some embodiments, the lipid nanoparticles have a molar ratio of 40:25:30:5 for cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids.

[0242] therapeutic use In some embodiments, the dry powder formulations described herein (e.g., reconstituteable dry powder formulations) or reconstituted dry powder formulations include any full-length mRNA. In some embodiments, the formulations described herein include mRNA encoding a drug or peptide or protein therapeutic agent suitable for the disclosure. In some embodiments, the formulations described herein include full-length mRNA encoding any peptide or polypeptide suitable for the disclosure. In some embodiments, the formulation is a dry powder formulation (e.g., a reconstituteable dry powder formulation). In some embodiments, the formulation is a reconstituted formulation (e.g., reconstituted from a reconstituteable dry powder formulation described herein). In some embodiments, the formulations described herein include other nucleic acids for in vivo administration, as discussed herein.

[0243] Exemplary non-limiting mRNAs suitable for use in dried powder and reconstituted LNP formulations are described herein.

[0244] Therapeutic proteins encoded by mRNA: Exemplary naturally occurring engineered proteins and peptides: In some embodiments, the formulations described herein include a full-length mRNA encoding any antibody suitable for the disclosure. In some embodiments, the formulations described herein include a full-length mRNA encoding any therapeutic protein suitable for the disclosure. In some embodiments, the formulations described herein include a full-length mRNA encoding any naturally occurring peptide suitable for the disclosure. In some embodiments, the formulations described herein include a full-length mRNA encoding any modified or non-natural peptide suitable for the disclosure. In some embodiments, the formulations described herein include a full-length mRNA encoding any peptide agent suitable for the disclosure.

[0245] In some embodiments, the mRNA encodes one or more naturally occurring peptides. In some embodiments, the mRNA encodes one or more modified or non-natural peptides.

[0246] In some embodiments, the peptide agent comprises glucose-dependent insulin-secreting polypeptides. A further example of a peptide agent is ellamipretide. Further examples of peptide agents include cyclotides (peptides characterized by their head-tail cyclized peptide backbone and interlock sequences of their disulfide bonds), for example, cyclotides having at least two disulfide bonds (and preferably cyclotides having three disulfide bonds).

[0247] In some embodiments, the peptide agent is GLP-1, amylin, amylin analogs, pramulintide, somatostatin analogs (e.g., octreotide, lanreotide, or pasireotide), goserelin (e.g., goserelin acetate), buserelin, peptide YY (PYY), PYY analogs, glatiramer (e.g., glatiramer acetate), leuprolide (e.g., leuprolide acetate), desmopressin (e.g., desmopressin acetate, especially desmopressin monoacetate trihydrate), teicoplanin, teravancin, bleomycin, lamopranin, decaplanin, bortezomib, cosyntropin, selmorelin, luteinizing agent. The following are selected from hormone-releasing hormone (LHRH), calcitonin (e.g., calcitonin-salmon), pentagastrin, neseritide, enfuvirtide, eptifivatide, cyclosporine, glucagon, biomycin, thyrotropin-releasing hormone (TRH), leucine-empharin, methionine-enkepharin, substance P, parathyroid hormone (PTH) fragments (e.g., teriparatide (PTH(1-34)), PTH(1-31) or PTH(2-34)), carfilzomib, icatiban, sirengitide, prostaglandin F2α receptor modulators (e.g., PDC31), and pharmaceutically acceptable salts thereof. The peptide agent is particularly preferably selected from semaglutide, liraglutide, teriparatide (PTH(1-34)), octreotide, leuprolide, and pharmaceutically acceptable salts thereof.

[0248] Exemplary proteins for tissue-specific delivery In some embodiments, the formulations described herein include full-length mRNA encoding any peptide or polypeptide for use in delivery to or treatment of lung or lung cells of a subject suitable for this disclosure.

[0249] In some embodiments, the formulations described herein include full-length mRNA encoding any peptide or polypeptide for use in delivery to or treatment of the liver or hepatocytes of a subject suitable for this disclosure.

[0250] In some embodiments, the formulations described herein include full-length mRNA encoding a protein associated with urea cycle disorders that is suitable for this disclosure.

[0251] In some embodiments, the formulations described herein include full-length mRNA encoding a protein associated with lysosomal storage disorders that is suitable for this disclosure.

[0252] In some embodiments, the formulations described herein include full-length mRNA encoding a glycogen storage disease-related protein suitable for this disclosure.

[0253] In some embodiments, the formulations described herein include full-length mRNA encoding a protein related to amino acid metabolism suitable for this disclosure.

[0254] In some embodiments, the formulations described herein include full-length mRNA encoding proteins related to lipid metabolism or fibrosis, which are suitable for this disclosure.

[0255] In some embodiments, the formulations described herein include full-length mRNA encoding a protein associated with methylmalonic acidemia that is suitable for this disclosure.

[0256] In some embodiments, the formulations described herein include full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of cardiovascular or cardiovascular cells of a subject suitable for this disclosure.

[0257] In some embodiments, the formulations described herein include full-length mRNA encoding any peptide or polypeptide for use in delivery to or treatment of muscle or muscle cells of a subject suitable for the present disclosure.

[0258] In some embodiments, the formulations described herein include full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of the nervous system or nervous system cells of a subject suitable for this disclosure.

[0259] In some embodiments, the formulations described herein include full-length mRNA encoding any peptide or polypeptide for use in delivery to or treatment of the eye or ophthalmic cells of a subject suitable for this disclosure.

[0260] Exemplary embodiments are described herein.

[0261] a. Liver In some embodiments, the present disclosure provides a formulation having full-length mRNA for delivery to the liver. In some embodiments, the dried powder formulation is reconstituted.

[0262] In some embodiments, the Disclosure provides formulations having full-length mRNA for delivery to the liver or formulations having full-length mRNA for the treatment of liver-related conditions. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the ATP7B protein, also known as Wilson's disease protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the porphobilinogen deaminase enzyme. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding one of the coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the human hemochromatosis (HFE) protein.

[0263] b.Lungs In some embodiments, the formulations described herein include full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of the lungs or cells of a subject suitable for this disclosure. Additional embodiments are described herein.

[0264] In some embodiments, the present disclosure provides formulations comprising full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of target lung or lung cells.

[0265] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a cystic fibrosis membrane conductance regulator (CFTR) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an ATP-binding cassette subfamily A member 3 protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a dynein axonemal intermediate chain 1 protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a dynein axonemal heavy chain 5 (DNAH5) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an alpha-1-antitrypsin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a forkhead box P3 (FOXP3) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding one or more surfactant proteins, for example, surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein. In some embodiments, a dry powder formulation containing trehalose is used for lung delivery. In some embodiments, formulations containing a sugar or sugar alcohol excipient having a particle size of less than 5 microns (e.g., trehalose) are suitable for pulmonary delivery.

[0266] c. heart In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of a target cardiovascular system or cardiovascular cells. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding vascular endothelial growth factor A protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding relaxin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding osteomorphogenetic protein-9 protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding osteomorphogenetic protein-2 receptor protein.

[0267] d. Muscle In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of a target muscle or muscle cell. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a dystrophin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a frataxin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of a target cardiac muscle or cardiomyocyte. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a protein that modulates one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a protein that modulates the Kv7.1 channel in muscle tissue or muscle cells. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a protein that modulates the Nav1.5 channel in muscle tissue or muscle cells.

[0268] e. nerve cells In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of the nervous system or nervous system cells of interest. For example, in some embodiments, the Disclosure provides formulations having full-length mRNA encoding the Survival Motor Neuron 1 protein. For example, in some embodiments, the Disclosure provides formulations having full-length mRNA encoding the Survival Motor Neuron 2 protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the frataxin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the ATP-binding cassette subfamily D member 1 (ABCD1) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding the CLN3 protein.

[0269] f. Bone marrow In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of blood or bone marrow or blood or bone marrow cells. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a beta-globin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a Bruton's tyrosine kinase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding one or more coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X.

[0270] g. kidney In some embodiments, the disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of a target kidney or kidney cells. In some embodiments, the disclosure provides formulations having full-length mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.

[0271] h.Eye In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of an eye or ophthalmic cells of interest. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a retinosuxin protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a retinal pigment epithelium-specific 65 kDa (RPE65) protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a 290 kDa centrosome protein (CEP290).

[0272] Vaccine proteins In some embodiments, the formulations described herein include a vaccine for a subject suitable for the present disclosure or a full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of target cells. Additional embodiments are described herein.

[0273] In some embodiments, the formulations described herein include a full-length mRNA encoding an antigen suitable for the disclosure (e.g., derived from an infectious agent such as a virus). In some embodiments, the formulations described herein include a full-length mRNA encoding an immunomodulatory agent suitable for the disclosure. In some embodiments, the formulations described herein include a full-length mRNA encoding an endonuclease suitable for the disclosure.

[0274] a. Exemplary proteins for cancer vaccines Cancer is considered an autoimmune disease, and cancer immunotherapy is at the forefront of research and development today. Cancer immunotherapy involves developing vaccines that strengthen the immune system to fight cancer antigens and eliminate tumors through activated cytotoxic T cells against these antigens. One class of cancer antigens is viral antigens associated with viruses that cause cancer, for example, Epstein-Barr virus (EBV) antigens such as EBV1 and EBV2 associated with lymphoma and nasopharyngeal cancer; human papillomavirus (HPV) antigens such as HPV16 associated with cervical cancer (CC); hepatitis B virus (HBV) antigens and hepatitis C virus (HCV) antigens associated with hepatocellular carcinoma (HCC); human T-lymphotropic virus type 1 (HTLV-1) associated with adult T-cell leukemia / lymphoma and human herpesvirus 8 (HHV-8) associated with Kaposi's sarcoma.

[0275] On the other hand, cancer cells express antigens that are not generally expressed by non-cancerous cells or tissues. Such antigens include, but are not limited to, epithelial tumor antigens (ETAs) found in breast cancer, RAS family member p-53 and other activated RAS antigens, ovarian cancer antigens BRCA1 and BRCA2, melanoma-associated antigens (MAGEs) found in malignant melanoma cells, BCR-ABL fusion gene products found in myeloid leukemia, BRAF antigens found in acute lymphoblastic leukemia, acute myeloplastic leukemia, cutaneous melanoma and colorectal cancer, epidermal growth factor receptor (EGFR) in non-small cell lung cancer, KRAS found in colorectal and non-small cell lung cancer, neuron-specific enolase found in neuroblastoma and non-small cell lung cancer, and NY-ESO found in neuroblastoma. 1. Melanoma-associated antigen (MART-1), recognized by T cells in melanoma, programmed death ligand 1 (PD-L1), prostate-specific antigen (PSA), urokinase activator (UPA), and plasminogen activator inhibitor (PAI-1), found in breast cancer and other cancers, are almost all mutated endogenous proteins. These endogenous cancer antigens are not presented by antigen-presenting cells (APCs) in the same way as viral antigens, namely in relation to the MHC-1 molecule that classifies antigens as exogenous antigens. However, cytotoxic T cells have the unique characteristic of being able to differentiate and identify mutated autoantigens and seek out and destroy cells that possess the mutated antigens. Therefore, the current goal of cancer immunotherapy is to achieve optimal activation of cytotoxic T cells against mutated antigens. Patient-specific mutations related to the patient's cancer can be mapped and used to generate vaccines, inducing the patient's own cytotoxic T cells to generate the immune response necessary to destroy tumor cells. mRNA vaccines may be a safe and cost-effective alternative to peptide vaccines to enable such personalized medicine. By using pan-genomic scanning and analysis of mutations present in cancer patients, mRNA encoding an antigen or epitope containing the mutation can be specifically designed. When this is administered in vivo, a translation product is produced on the cell surface. This will trigger an immune response against the mutated antigen. In this process, cytotoxic T cells attack tumor cells that essentially express the mutated antigen.Methods and protocols involved in performing pan-genome sequencing analysis, mutation analysis, epitope mapping and analysis, and designing peptides suitable for vaccination are known to those skilled in the art.

[0276] This approach can also be used to identify a patient's dominant and subdominant antigens. In both chronic infections and cancer, certain antigens have been observed to play a dominant role in generating the initial immune response. However, shortly thereafter, resistance to such dominant antigens is established, thereby reducing the immune response. Genomic analysis and identification of antigens that did not show an initial dominant antigenic response (often called subdominant antigens) can then be used to generate a newly reactivated immune response.

[0277] One advantage of mRNA vaccines over peptide vaccines is that mRNA vaccines bypass HLA compatibility in the recipient host.

[0278] A synthetic approach to mRNA vaccine design allows for scanning the pathogen proteome for antigenic signatures with vaccine potential. (Proteome databases can be evaluated using the Uniprot Consortium, uniprot.org / ). This may be effective against novel pathogens such as the Zika virus. This type of reverse vaccinology has been used to identify numerous novel peptide vaccine candidates. By combining genomics and proteomics, novel peptide vaccines have also been identified from Helicobacter pylori and Mycobacterium tuberculosis (see, e.g., Etz et al., PNAS. 2002, 99(10) 6573-6578). Whether a potential antigen candidate can generate a successful immune response can be verified by properly expressing a library of potential antigens using various forms of cell surface display and subjecting them to opsonization and antibody binding tests. Examples of useful databases for vaccine antigen development include: ImMunoGeneTics Information System (URL: imgt.org); Epitome Database (URL: rostlab.org / services / epitome); Immune Epitope Database and Analysis Resource, iedb.org; Immunet Database, immunoet.cn / ced / index.php; HIV Database: hiv.lanl.gov / content / immunology (for immunogenetics and immunoinformatics).

[0279] Therefore, from the above considerations, it is clear that enrichment of mRNA vaccine delivery to lymph nodes can lead to vaccine access to both activated lymphocytes and naive lymphocytes for lymphoid proliferation and antigen-specific T and B cell generation. This localization of antigen activation is also less toxic, in contrast to broader immune activation.

[0280] b. Exemplary proteins for vaccines against infectious diseases mRNA vaccines offer many advantages over current cell-based vaccines that use live, attenuated, or dead pathogens or toxoid vaccines. The dried powder formulations of this disclosure can be used to deliver mRNA for vaccination. In addition to safety, mRNA vaccines are cost-effective and offer a flexible design platform. mRNA encoding antigens can be directed to induce a specific immune response and can therefore be applied to the development of a wide range of therapeutic and prophylactic mRNA vaccines for a diverse range of diseases, including infectious diseases and cancer.

[0281] COVID-19, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), measles, avian influenza, chikungunya virus, Ebola virus disease (EVD), and Zika virus disease have brought a new focus to infectious diseases. Continued readiness and preparedness are needed to address outbreaks of infectious diseases, including new and re-emerging threats.

[0282] Vaccine candidates are well-established against numerous infectious pathogens. Typically, a vaccine is an agent that at least partially mimics a disease-causing agent, thereby inducing an immune response by a mammalian host. Generally, vaccines are biological agents such as heat-sterilized, irradiated, or other attenuated pathogens, attenuated live microorganisms, protein or peptide antigens, conjugate antigens, toxins, or microbial surface proteins or fragments thereof. However, mRNA encoding protein or peptide antigens is a safe and effective method for inducing an immune response to disease. As discussed above, mRNA can be effectively delivered to be expressed in vivo by encapsulating it in liposomes containing suitable lipids, which will be discussed in later sections. Thus, this mRNA encoding an antigenic peptide or protein can be used to produce a vaccine in vivo. The immune response produced by the mammalian host against the vaccine component is intended to protect the host from subsequent attacks by the pathogen, as the host's immune system is then primed for attack by the pathogen. In other words, the host system has immunological memory of the pathogen (a component of the adaptive immune response). This process is known as prophylactic vaccination. Furthermore, vaccines can enhance the host's immune system in pre-existing infections, for example, by redirecting the immune response to novel, less recognizable microbial antigens (subdominant antigens) that lead to a robust immune response resulting in the elimination of the pathogen. This type of vaccine response can be classified as therapeutic vaccination.

[0283] The immune response to pathogens can be divided into several stages. Firstly, the encounter between the human body (or mammalian system) and a new pathogen, particularly through contact via exposed surfaces such as the skin or the internal mucosal surfaces of the respiratory, gastrointestinal, and urogenital tracts, results in a nonspecific innate immune response through the activation of pattern recognition molecules. Pattern recognition molecules include various germline coding receptors specialized in distinguishing between microbial cell surfaces and host cell surfaces, or between infected cells and normal cells. Phagocytes (monocytes, macrophages, and dendritic cells) express pattern recognition molecules on their surfaces and are primarily responsible for the recognition, death, and elimination of pathogens in the innate immune response. In this process, phagocytes process antigens and present them to circulating lymphocytes to generate a more specific antigen-targeted immune response, also known as an adaptive immune response. At this stage, activated lymphocytes mature into antigen-specific T cells in the lymph nodes, expressing receptors for antigen recognition so that effector cytotoxic T cells recognize and kill cells expressing the antigen when it is associated with a second set of cell surface molecules, major histocompatibility complex molecules or MHC. Helper T cells activate the system to generate T cell memory and a humoral immune response. The humoral immune response includes antibody-secreting B cells produced by clonal expression and differentiation over several days, during which time the innate immune system continues to function. Clonal proliferation of cytotoxic T cells occurs rapidly in lymphoid organs such as lymph nodes and is enhanced by exposure to the antigen. Activated T cells produce several cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are considered characteristic of T cell activation. Finally, antigen-specific T cells, and then antibodies, are released into the bloodstream and recruited to the site of infection. A successful vaccine produces a rapid and robust cytotoxic T cell response, a strong antibody response, and persistent immunological memory.

[0284] Exemplary cell maintenance proteins In some embodiments, the disclosure provides formulations having full-length mRNA encoding proteins related to lipid metabolism or fibrous disorders. In some embodiments, the disclosure provides formulations having full-length mRNA encoding mTOR inhibitors. In some embodiments, the disclosure provides formulations having full-length mRNA encoding the ATPase phospholipid transporter 8B1 (ATP8B1) protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding one or more NF-kappa B inhibitors, such as one or more of I-kappa B alpha, interferon-related developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In some embodiments, the disclosure provides formulations having full-length mRNA encoding PPAR-gamma proteins or active variants.

[0285] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use as a vaccine for a target or for delivery to cells of a target or for the treatment thereof. For example, in some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen from an infectious agent such as a virus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from influenza virus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from respiratory syncytial virus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from rabies virus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from cytomegalovirus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from rotavirus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from hepatitis viruses such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from human papillomavirus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from human metapneumovirus. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen derived from malaria virus.In some embodiments, the disclosure provides formulations having full-length mRNA encoding an antigen derived from the Zika virus. In some embodiments, the disclosure provides formulations having full-length mRNA encoding an antigen derived from the Chikungunya virus.

[0286] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an antigen associated with or identified from cancer cells of the subject. In some embodiments, the Disclosure provides formulations for providing personalized cancer vaccines, i.e., formulations having full-length mRNA encoding an antigen determined from cancer cells of the subject itself.

[0287] In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody. In some embodiments, the antibody may be a bispecific antibody. In some embodiments, the antibody may be part of a fusion protein. In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody against OX40. In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody against VEGF. In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody against tissue necrosis factor alpha. In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody against CD3. In some embodiments, the disclosure provides a formulation having a full-length mRNA encoding an antibody against CD19.

[0288] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an immunomodulatory agent. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding interleukin-12. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding interleukin-23. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding interleukin-36 gamma. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding constitutively active variants of one or more stimulants of the interferon gene (STING) protein.

[0289] Polypeptides involved in the CRISPR / Cas9 system: In some embodiments, the Disclosure provides compositions having full-length mRNA encoding an endonuclease. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding an RNA-guided DNA endonuclease protein, such as the Cas9 protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a meganuclease protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a transcription activator-like effector nuclease protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a zinc finger nuclease protein.

[0290] Exemplary proteins found in various organelles In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding secretory proteins. In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding nucleoproteins. In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding metabolite proteins. In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding cytoplasmic proteins. In some embodiments, the disclosure provides compositions comprising full-length mRNA encoding membrane proteins. In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding mitochondrial proteins. In some embodiments, the disclosure provides formulations comprising full-length mRNA encoding lysosomal proteins. In some embodiments, the mRNA encodes cytosolic proteins. In some embodiments, the mRNA encodes proteins related to the actin cytoskeleton. In some embodiments, the mRNA encodes proteins related to the plasma membrane.

[0291] Metabolic metabolic proteins and catabolic proteins In some embodiments, the Disclosure provides formulations having full-length mRNA encoding a peptide or polypeptide for use in delivery to or treatment of a target liver or hepatocyte. Such peptides and polypeptides may include those related to urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery to or treatment of the liver or hepatocyte with enriched full-length mRNA would be beneficial.

[0292] In some embodiments, the disclosure provides formulations having full-length mRNA encoding proteins associated with urea cycle disorders. In some embodiments, the disclosure provides formulations having full-length mRNA encoding ornithine transcarbamylase (OTC) protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding arginosuccinate synthase 1 protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding carbamoyl phosphate synthase 1 protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding arginosuccinate lyase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding arginase protein.

[0293] In some embodiments, the disclosure provides formulations having full-length mRNA encoding proteins associated with methylmalonic acidemia. For example, in some embodiments, the disclosure provides formulations having full-length mRNA encoding methylmalonyl-CoA mutase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding methylmalonyl-CoA epimerase protein.

[0294] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding proteins associated with lysosomal storage disorders. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding alpha-galactosidase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding glucocerebrosidase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding iduronate-2-sulfatase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding iduronidase protein. In some embodiments, the Disclosure provides therapeutic formulations having full-length mRNA encoding N-acetyl-alpha-D-glucosaminidase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding heparan-N-sulfatase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding galactosamine-6-sulfatase protein. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding beta-galactosidase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding a lysosomal lipase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding the transcription factor EB (TFEB).

[0295] In some embodiments, the disclosure provides formulations having full-length mRNA encoding proteins associated with glycogen storage disorders. In some embodiments, the disclosure provides formulations having full-length mRNA encoding acid alpha-glucosidase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding glucose-6-phosphatase (G6PC) protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding hepatic glycogen phosphorylase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding muscle phosphoglycerate mutase protein. In some embodiments, the disclosure provides formulations having full-length mRNA encoding glycogen debranchase.

[0296] In some embodiments, the Disclosure provides formulations having full-length mRNA encoding proteins related to amino acid metabolism. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding phenylalanine hydroxylase enzyme. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding glutaryl-CoA dehydrogenase enzyme. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding propionyl-CoA carboxylase enzyme. In some embodiments, the Disclosure provides formulations having full-length mRNA encoding oxalase alanine-glyoxylate aminotransferase enzyme.

[0297] In some embodiments, the disclosure provides formulations containing nucleic acids, such as plasmids, viral vectors, antisense nucleic acids, siRNA, microRNA, ribozymes, antagomirs, aptamers, CRISPR nucleic acids (e.g., guide RNA, crRNA, or tracrRNA), nucleic acids for gene therapy, nucleic acids for DNA editing, probes, or nucleases and / or other oligonucleotides that are susceptible to degradation under harsh environmental conditions (e.g., pH) including therapeutic nucleic acids.

[0298] Delivery method The compositions described herein may be administered as a dry powder formulation or, after reconstitution, according to various methods known in the art. For example, after reconstitution, the pharmaceutical formulations of the Disclosure may be administered topically rather than systemically, for example, by direct injection of a pharmaceutical formulation, including a sustained-release formulation, into a target tissue.

[0299] For example, the dried powder formulations and / or reconstituted dried powder formulations of the present invention may be administered and prescribed in accordance with existing medical practices, taking into consideration the clinical condition of the subject, the properties of the encapsulation material, the site and method of administration, the plan of administration, the age, sex, weight of the subject, and other factors relevant to a clinician with ordinary art in the art.

[0300] Preferred routes of delivery of the dried powder formulations and / or reconstituted dried powder formulations disclosed herein include, for example, oral, rectal, vaginal, transmucosal, or intestinal delivery; parenteral delivery (including intramuscular, subcutaneous, intrathecal injection, and intrathecal, intraventricular, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection or infusion).

[0301] The dried powder formulations and / or reconstituted dried powder formulations of the present invention may be administered topically rather than systemically. For example, the reconstituted dried powder formulation may be administered by direct injection or infusion of the pharmaceutical composition into target tissue, preferably in a depot formulation or sustained-release formulation, so that contact between target cells and constituent lipid nanoparticles may be further enhanced. Topical delivery may be achieved in various ways depending on the target tissue. For example, an aerosol containing the composition of the present invention may be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention may be injected, for example, into a site of injury, symptom onset of disease, or pain; the composition may be provided in lozenges for oral, tracheal, or esophageal application; it may be supplied in the form of a liquid, tablet, or capsule for administration to the stomach or intestine, or in the form of a suppository for rectal or vaginal application; or it may even be delivered in the eye, using a cream, eye drops, or even injection. Formulations of the present invention that form complexes with therapeutic branches or ligands may further be administered surgically, for example, in conjunction with a polymer or other structure or substance that can enable the composition to diffuse from the implantation site to the surrounding site. Alternatively, such compositions may be surgically applied without the use of polymers or supports.

[0302] Further exemplary, non-limiting delivery modes are described herein.

[0303] i. Targeted delivery Local delivery can be achieved in various ways depending on the target tissue. Exemplary tissues to which delivered mRNA may be delivered and / or expressed include, but are not limited to, the lungs, liver, kidneys, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In some embodiments, the liver is targeted. For example, the aerosol-containing compositions of this disclosure can be inhaled (for nasal, tracheal, or bronchial delivery).

[0304] ii. Delivery to the airway In some embodiments, the compositions of the Disclosure may be delivered using a metered-dose inhaler. In some embodiments, the compositions of the Disclosure may be reconstituted and sprayed for delivery. In some embodiments, the compositions of the Disclosure may be injected into a site of injury, disease symptom onset, or pain. In some embodiments, the compositions of the Disclosure may be provided for oral, tracheal, or esophageal application.

[0305] In some embodiments, the formulations of the present disclosure are reconstituted into a liquid solution and sprayed for delivery. Spraying can be achieved by any nebulizer known in the art. A nebulizer converts the liquid into a mist so that it can be more easily inhaled into the lungs. Nebulizers are effective for infants, children and adults. Nebulizers can spray large volumes of inhaled medication. Typically, a nebulizer for use with the present disclosure includes a removable mouthpiece.

[0306] Treatments for liver and lung diseases have been developed by delivering synthetic mRNA encoding deficient and / or non-functional proteins to corresponding organ cells via intravenous and inhalation routes, respectively.

[0307] In particular, in the case of pulmonary delivery, the particles of the formulation affect the distribution and deposition of aerosols within the respiratory system. Often, particle deposition in larger inductive airways is preferred for effective absorption and distribution of the therapeutic component. Aerosols of very fine particles, e.g., particles with a diameter of less than 1 micrometer, can be deposited peripherally for effective absorption by specific cells in the lung, such as smooth muscle, for the active pharmaceutical component that functions as a bronchodilator. In some embodiments, formulations containing trehalose are used for pulmonary delivery. In some embodiments, formulations containing sugar or sugar alcohol excipients (e.g., trehalose) with a particle size of less than 5 microns are suitable for pulmonary delivery.

[0308] iii. Tablets and granules In some embodiments, the compositions of the present disclosure may be supplied in the form of a reconstituted liquid, powder, tablet, granule, implant, or capsule for administration into the stomach or intestine. In some embodiments, the pharmaceutical composition is in the form of a tablet with an enteric coating.

[0309] In some embodiments, the enteric coating includes: hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate, cellulose phthalate acetate, cellulose trimellitate acetate, hydroxypropylmethylcellulose phthalate, cellulose ester-ether phthalate, hydroxypropylcellulose phthalate, alkali salts of cellulose phthalate acetate, alkaline earth salts of cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, cellulose phthalate acetate, sodium carboxymethylcellulose, acrylic acid polymers and copolymers, terpolymers of ethyl acrylate / methyl methacrylate / ethyltrimethylammonium chloride, copolymers of methacrylate / ethyl acrylate, copolymers of methacrylate / methyl methacrylate, polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, copolymers of vinyl crotonic acid acetate; shellac, ammonia-treated shellac, shellac-acetyl alcohol, or shellac n-butyl stearate.

[0310] In some embodiments, tablets are manufactured using fillers-binders for direct compression and to promote cohesiveness. Some exemplary binders include polyvinylpyrrolidone (PVP), methylcellulose, hydroxypropyl methylcellulose (HPMC), polymethacrylate, sodium carboxymethylcellulose, polyethylene glycol (PEG), and methylcellulose, sucrose, acacia, methylcellulose, liquid glucose, tragacanth, ethylcellulose, gelatin, starch paste, hydroxypropylcellulose, pregelatinized starch, sodium carboxymethylcellulose, alginic acid, polyvinyl alcohol, and polymethacrylate.

[0311] iv. Other routes of administration In some embodiments, the compositions of the present disclosure may be supplied for rectal or vaginal application. In some embodiments, the compositions of the present disclosure may be delivered to the eye as drops or even as intravitreous or intraocular injection.

[0312] In some embodiments, the compositions of this disclosure can be used via any parenteral and mucosal routes. In some embodiments, liquid formulations can be administered, for example, by infusion, perfusion, administration using a pen syringe, cartridge system needle array or patch, and / or administration by a catheter system. In some embodiments, administration is by subcutaneous injection, intradermal injection, subcutaneous injection, intramuscular injection, or topical administration. There are many common forms of topical medications, such as lotions, gels, patches, and powders, creams, and ointments. In some embodiments, parenteral administration includes oral, sublingual, palatal, gingival, nasal, vaginal, cervical, rectal, or transdermal administration. In some embodiments, parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, and intraventricular administration.

[0313] In some embodiments, the method of administration may further include oral administration. In some examples, oral administration may include mouthwash, rinsing, oral rinse, mouth bath, etc. For example, cervical and / or vaginal mucosal administration may be via a solution, gel, suspension, cream, ointment, foam, pessary, or tablet. In one embodiment, the reconstituted composition may be administered to the target cervical and / or vaginal mucosa. Cervical and / or vaginal mucosal administration may be via a solution, gel, suspension, cream, ointment, foam, pessary, or tablet.

[0314] In some embodiments, the formulation or reconstituted formulation is suitable for mucosal delivery. In some embodiments, the formulation is suitable for oral delivery. In some embodiments, the formulation is suitable for sublingual delivery. In some embodiments, the formulation is suitable for intranasal delivery. In some embodiments, the formulation is suitable for oral delivery. In some embodiments, the formulation is suitable for intramuscular delivery. In some embodiments, the formulation is suitable for intravenous delivery. In some embodiments, the formulation is suitable for subcutaneous delivery.

[0315] A First Representative Embodiment of the Present Disclosure 1. A dried powder formulation for reconstitution, (a) an excipient which is sucrose or trehalose, (b) A lipid nanoparticle (LNP) containing messenger RNA (mRNA) encapsulated by one or more lipids, A dried powder formulation wherein the w / w ratio of the excipient (a) to the total lipids in the LNP (b) is at least about 5. 2. The dried powder formulation according to numbered embodiment 1, wherein the excipient is sucrose. 3. The dried powder formulation according to numbered embodiment 1, wherein the excipient is trehalose. 4. A dried powder formulation according to any one of the numbered embodiments 1 to 3, wherein the w / w ratio of the excipient to the total lipids is at least about 5.6, 11, or 15. 5. The dried powder formulation according to numbered embodiment 4, wherein the w / w ratio of the excipient to the total lipids is at least about 11.1 or at least about 15.6. 6. A dried powder formulation according to any one of the numbered embodiments 1 to 5, comprising an LNP containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. 7. A dried powder formulation for reconstitution, (a) an excipient which is a sugar or sugar alcohol, (b) A lipid nanoparticle (LNP) containing messenger RNA (mRNA) encapsulated by one or more lipids, A dried powder formulation wherein the w / w ratio of the excipient (a) to the total lipids in the LNP (b) is at least about 5. 8. The dried powder formulation according to numbered embodiment 7, wherein the sugar or sugar alcohol is selected from sucrose, mannitol, xylitol, lactose, and trehalose. 9. The dried powder formulation according to the numbered embodiment 7, wherein the sugar or sugar alcohol is trehalose. 10. The dried powder formulation according to the numbered embodiment 7, wherein the sugar or sugar alcohol is sucrose. 11. A dried powder formulation according to any one of the numbered embodiments 7 to 10, wherein the w / w ratio of sugar or sugar alcohol to total lipids is at least about 5.6, 11, or 15. 12. The dried powder formulation according to numbered embodiment 7, wherein the w / w ratio of sugar or sugar alcohol to total lipids is at least about 11.1 or at least about 15.6. 13. A dried powder formulation according to any one of the numbered embodiments 7 to 12, comprising an LNP containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. 14. A dried powder formulation according to any one of the numbered embodiments 1 to 13, wherein the particle size of the reconstituted dried powder formulation is less than 120 nm. 15. A dried powder formulation according to any one of the numbered embodiments 1 to 14, wherein the encapsulation rate is greater than 60%. 16. A dried powder formulation according to any one of the numbered embodiments 1 to 15, wherein the particle size of the dried powder is less than 5 μm. 17. A dried powder formulation according to any one of the numbered embodiments 1 to 16, wherein the formulation is stable after long-term storage. 18. A dried powder formulation according to numbered embodiment 17, wherein the mRNA maintains 80% or more integrity after storage at 4°C for at least 6 months. 19. A dried powder formulation according to numbered embodiment 17, wherein the mRNA maintains 80% or more integrity for up to one year. 20. A dried powder formulation according to numbered embodiment 17, wherein the mRNA maintains 80% or more integrity for at least one year. 21. A dried powder formulation according to any one of the numbered embodiments 1 to 20, wherein the mRNA encodes a therapeutic protein. 22. A dried powder formulation according to any one of the numbered embodiments 1 to 21, wherein the mRNA encodes an antigen. 23. A dried powder formulation according to any one of the numbered embodiments 1 to 22, wherein the formulation is suitable for use as a vaccine. 24. A dried powder formulation according to any one of the numbered embodiments 1 to 23, wherein the formulation is suitable for parenteral delivery. 25. The reconstituted dried powder formulation according to numbered embodiment 24, wherein the formulation is suitable for intramuscular, intravenous, or subcutaneous delivery. 26. A reconstituted dried powder formulation according to any one of the numbered embodiments 1 to 25, wherein the formulation is suitable for mucosal delivery. 27. The reconstituted dried powder formulation according to numbered embodiment 26, wherein the formulation is suitable for oral, sublingual, or intranasal delivery. 28. A method for delivering mRNA in vivo, comprising administering a reconstituted dried powder formulation described in any one of the numbered embodiments 1 to 27 to a subject requiring the delivery of mRNA. 29. A method for treating a disease or disorder in a subject, comprising administering a reconstituted dried powder formulation described in any one of the numbered embodiments 1 to 28 to the subject. 30. The method according to numbered embodiment 28 or 29, wherein the reconstituted dried powder formulation is administered subcutaneously, intravenously, or intramuscularly. 31. A method for preparing a dried powder formulation for reconstitution, (a) Combining a mixture containing lipid nanoparticles (LNPs) for encapsulating mRNA with an ethanol solution, wherein the ethanol solution contains sucrose or trehalose at a concentration of approximately 1-11% (w / v), (b) A method comprising spray-drying a mixture to obtain a dry powder formulation. 32. A method for preparing a reconstituted dried powder formulation, (a) To provide a dried powder formulation prepared according to the numbered embodiment 31, (b) A method comprising reconstituting a dried powder formulation in water or a buffer solution to obtain a reconstituted dried powder formulation. 33. The method according to the numbered embodiment 32, wherein the reconstructed LNP has a diameter of approximately 100 nm. 34. The method according to the numbered embodiment 31 or 32, wherein the ratio (w / w) of sucrose or trehalose to total lipids is at least about 5, at least about 11, or at least about 15. 35. The method according to numbered embodiment 31 or 32, wherein the concentration of sucrose or trehalose is greater than 2% (w / v), greater than 5% (w / v), or greater than 7% (w / v) after reconstitution. 36. The method according to any one of the numbered embodiments 31 to 35, wherein the sugar concentration after reconstitution is approximately 2 (w / v) to approximately 10% (w / v). 37. The method according to any one of the numbered embodiments 31 to 36, wherein the particle size of the reconstituted dried powder formulation is less than 120 nm. 38. The method according to any one of the numbered embodiments 31 to 37, wherein the particle size of the reconstituted LNP is 80 to 120 nm. 39. The method according to any one of the numbered embodiments 31 to 38, wherein the particle size of the reconstituted LNP is 80 to 115 nm.

[0316] A Second Representative Embodiment of the Present Disclosure 1. A dried powder formulation for reconstitution, (a) an excipient which is a sugar or sugar alcohol, (b) A lipid nanoparticle (LNP) containing messenger RNA (mRNA) encapsulated by one or more lipids, A dried powder formulation wherein the w / w ratio of the excipient (a) to the total lipids in the LNP (b) is at least about 5. 2. Sugar or sugar alcohol, (a) Selected from sucrose, mannitol, xylitol, lactose, and trehalose, or (b) It is trehalose, or (c) The dried powder formulation according to Embodiment 1, wherein the dry powder formulation is sucrose. 3. The dried powder formulation according to Embodiment 1 or 2, wherein the w / w ratio of sugar or sugar alcohol to total lipids is at least about 5.6, 11, or 15, and optionally, the w / w ratio of sugar or sugar alcohol to total lipids is at least about 11.1 or at least about 15.6. 4. A dried powder formulation according to any one of Embodiments 1 to 3, comprising LNP containing one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. 5. A dried powder formulation according to any one of Embodiments 1 to 4, wherein the particle size of the reconstituted dried powder formulation is less than 120 nm. 6. A dried powder formulation according to any one of Embodiments 1 to 5, wherein the encapsulation rate is greater than 60%. 7. A dried powder formulation according to any one of Embodiments 1 to 6, wherein the particle size of the dried powder formulation is less than 5 μm. 8. The formulation is stable after long-term storage, and the mRNA is selectively selected. (a) 80% or more after storage at 4°C for at least 6 months; or (b) 80% or more for a maximum of one year; or (c) A dried powder formulation according to any one of Embodiments 1 to 7, which maintains 80% or more integrity for at least one year. 9. A dried powder formulation according to any one of Embodiments 1 to 8, wherein the mRNA encodes a therapeutic protein. 10. A dried powder formulation according to any one of Embodiments 1 to 9, wherein the mRNA encodes an antigen. 11. A dried powder formulation according to any one of Embodiments 1 to 10, wherein the formulation is suitable for use as a vaccine. 12. The formulation, (a) Suitable for parenteral delivery, and optionally, the formulation is suitable for intramuscular, intravenous, or subcutaneous delivery; and / or (b) A dried powder formulation according to any one of Embodiments 1 to 11, which is suitable for mucosal delivery, and optionally, the formulation is suitable for oral, sublingual, or intranasal delivery. 13. A method for delivering mRNA in vivo, comprising administering a reconstituted dried powder formulation described in any one of Embodiments 1 to 12 to a subject requiring it, wherein the reconstituted dried powder formulation is optionally administered subcutaneously, intravenously, or intramuscularly. 14. A method for treating a disease or disorder in a subject, wherein the reconstituted dried powder formulation described in any one of Embodiments 1 to 13 is administered to the subject, thereby optionally administering the reconstituted dried powder formulation subcutaneously, intravenously, or intramuscularly. 15. A method for preparing a dried powder formulation for reconstitution, (a) Combining a mixture containing lipid nanoparticles (LNPs) for encapsulating mRNA with an ethanol solution, wherein the ethanol solution contains sucrose or trehalose at a concentration of approximately 1-11% (w / v), (b) spray-drying the mixture to obtain a dry powder formulation, optionally comprising, (i) The method is (c) To provide a dried powder formulation, (d) Reconstitute the dried powder formulation in water or buffer solution to obtain a reconstituted dried powder formulation, further comprising optionally having a reconstituted LNP having a diameter of about 100 nm, and / or (ii) The ratio of sucrose or trehalose to total lipids (w / w) is at least about 5, at least about 11, or at least about 15, or (iii) The concentration of sucrose or trehalose after reconstitution is greater than 2%(w / v), greater than 5%(w / v), or greater than 7%(w / v), and / or (iv) The sugar concentration after reconstitution is approximately 2(w / v) to approximately 10%(w / v), and / or (v) The particle size of the reconstituted dried powder formulation is less than 120 nm, and / or (vi) The particle size of the reconstituted LNP is 80-120 nm, and / or (vii) A method in which the particle size of the reconstructed LNP is 80-115 nm. [Examples]

[0317] While specific compounds, compositions, and methods described herein are described in a particular manner according to specific embodiments, the following examples are for illustrative purposes only and are not intended to limit the disclosure.

[0318] Example 1 - Preparation of mRNA-LNP-based DPP The objective of this experiment was to synthesize mRNA-LNP-based dry powder products (DPPs) containing various sugar alcohol excipients.

[0319] The target mRNA was synthesized by in vitro transcription using RNA polymerase with a plasmid DNA template encoding the gene, using unmodified nucleotides. Subsequently, a 5' cap structure (Cap 1) and a 3' poly(A) tail were added. An ethanol solution of a mixture of lipids (ionic lipids, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipids) was combined with an aqueous buffer solution of the target mRNA under controlled conditions and at an acidic pH to obtain a homogeneous mRNA-LNP suspension. The mRNA-LNP suspension was formulated into final dilutions containing different sugar-alcohols and sugar excipients at desired concentrations in 20% ethanol during ultrafiltration and dialysfiltration. The resulting mRNA-LNP suspension was spray-dried in a Buchi-290 spray dryer using the parameters shown in Table 1. Various excipient amounts, as shown in Tables 2 and 3, were evaluated to identify the optimal excipient-to-total lipid ratio for producing a stable reconstituted product.

[0320] Several mRNA-LNP-containing dried powder products were prepared using various sugar alcohol excipients.

[0321] [Table 1]

[0322] [Table 2]

[0323] [Table 3]

[0324] Example 2 - Effects of various sugars and sugar alcohol excipients This experiment aimed to define the best sugar or sugar alcohol excipients for mRNA-LNP-based DPPs. All physical properties, including dry powder sprayability, particle size, and possibly acidity index, were analyzed and summarized.

[0325] i. Xylitol and lactose: As shown in Figures 1A and 1B, the formulations using xylitol (Figure 1A) and lactose (Figure 1B) did not spray dry sufficiently at the maximum excipient amount. The liquid formulations adhered to the cyclone separator, and the dried powder product was not collected in the collection container.

[0326] ii. Mannitol: The dry powder properties were excellent, but DPP was not reconstituted. The DPP yield was approximately 35% at mannitol / lipid weight ratios of 5.6 or higher, and increased with increasing mannitol / lipid ratio. Large aggregates were detected in the reconstituted dry powder product under 400x magnification, even when using the highest mannitol / lipid weight ratio of 15.6 (i.e., 7% mannitol (w / v); Figure 2A). Table 4 shows the dry powder properties using mannitol as a sugar excipient, as well as the composition and properties after reconstitution. Figure 2A shows aggregates observed in the reconstituted dry powder using 7% mannitol (w / v) as an excipient under 400x magnification compared to water alone. Figure 2B shows the appearance of the reconstituted dry powder using different amounts of mannitol as an excipient (left: 7% mannitol (w / v); center: 5% mannitol (w / v); right: 2.5% mannitol (w / v)).

[0327] iii. Sucrose: Although the DPP particle size was larger, the product was well reconstituted at a sucrose-...

Claims

1. A dried powder formulation for reconstitution, (a) an excipient which is sucrose or trehalose, (b) Lipid nanoparticles (LNPs) containing messenger RNA (mRNA) encapsulated by one or more lipids, Includes, A dried powder formulation wherein the excipient in (a) and the total lipids in the LNP in (b) have a weight ratio of at least about 5.

2. The dried powder formulation according to claim 1, wherein the excipient is sucrose.

3. The dried powder formulation according to claim 1, wherein the excipient is trehalose.

4. The dried powder formulation according to any one of claims 1 to 3, wherein the weight ratio of the excipient to the total lipids in the LNP is at least about 5.6, at least about 11, or at least about 15.

5. The dried powder formulation according to claim 4, wherein the weight ratio of the excipient to the total lipids is at least about 11.1 or at least about 15.

6.

6. The dried powder formulation according to any one of claims 1 to 5, wherein the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

7. A dried powder formulation for reconstitution, (a) an excipient which is a sugar or sugar alcohol, (b) Lipid nanoparticles (LNPs) containing messenger RNA (mRNA) encapsulated by one or more lipids, Includes, A dried powder formulation wherein the weight ratio of the excipient in (a) to the total lipids in the LNP in (b) is at least about 5.

8. The dried powder formulation according to claim 7, wherein the sugar or sugar alcohol is sucrose, mannitol, xylitol, lactose, or trehalose.

9. The dried powder formulation according to claim 7, wherein the sugar or sugar alcohol is trehalose.

10. The dried powder formulation according to claim 7, wherein the sugar or sugar alcohol is sucrose.

11. The dried powder formulation according to any one of claims 7 to 10, wherein the weight ratio of the sugar or sugar alcohol to the total lipids in the LNP is at least about 5.6, at least about 11, or at least about 15.

12. The dried powder formulation according to claim 7, wherein the weight ratio of the sugar or sugar alcohol to the total lipids in the LNP is at least about 11.1 or at least about 15.

6.

13. The dried powder formulation according to any one of claims 7 to 12, wherein the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

14. The dried powder formulation according to any one of claims 1 to 13, wherein the particle size of the reconstituted dried powder formulation is less than approximately 120 nm.

15. A dried powder formulation according to any one of claims 1 to 14, wherein the encapsulation rate is more than approximately 60%.

16. The dried powder formulation according to any one of claims 1 to 15, wherein the particle size of the dried powder is less than about 5 μm.

17. The dried powder formulation according to any one of claims 1 to 16, wherein the dried powder formulation is stable after long-term storage, for example at 2 to 8°C (for example at 4°C).

18. The dried powder formulation according to claim 17, wherein the mRNA maintains about 80% or more of its integrity after being stored, for example, at 2 to 8°C (for example, 4°C) for at least about 6 months.

19. The dried powder formulation according to claim 18, wherein the mRNA maintains about 80% or more of its integrity after being stored at, for example, 2 to 8°C (for example, 4°C) for up to about one year.

20. The dried powder formulation according to claim 19, wherein the mRNA maintains approximately 80% or more integrity when its mRNA integrity is measured after being optionally stored at 2 to 8°C (e.g., 4°C) for at least one year.

21. The dried powder formulation according to any one of claims 1 to 20, wherein the mRNA encodes a therapeutic protein.

22. The dried powder formulation according to any one of claims 1 to 21, wherein the mRNA encodes an antigen.

23. The dried powder formulation according to any one of claims 1 to 22, wherein the formulation is suitable for use as a vaccine.

24. The dried powder formulation according to any one of claims 1 to 23, wherein the formulation is suitable for parenteral delivery when reconstituted.

25. The dried powder formulation according to claim 24, wherein the formulation, when reconstituted, is suitable for intramuscular, intravenous, or subcutaneous delivery.

26. The dried powder formulation according to any one of claims 1 to 25, wherein the formulation is suitable for mucosal delivery when reconstituted.

27. The dried powder formulation according to claim 26, wherein the formulation, when reconstituted, is suitable for oral, sublingual, or intranasal delivery.

28. A method for delivering mRNA in vivo, comprising administering a reconstituted form of a dried powder formulation according to any one of claims 1 to 27 to a subject requiring the delivery of mRNA.

29. A method for treating a disease or disorder in a subject, comprising administering to the subject a reconstituted form of a dried powder formulation according to any one of claims 1 to 27.

30. The method according to claim 28 or 29, wherein the reconstituted form of the dried powder formulation is administered subcutaneously, intravenously, or intramuscularly.

31. A method for preparing a dried powder formulation for reconstitution, (a) Combining a first mixture containing lipid nanoparticles (LNPs) with an ethanol solution to obtain a second mixture, wherein the LNPs contain messenger RNA (mRNA) encapsulated by one or more lipids, and the ethanol solution contains an excipient that is sucrose or trehalose at a concentration of about 1-11% (w / v), (b) The second mixture is spray-dried to obtain the dried powder formulation, Methods that include...

32. A method for preparing a reconstituted dried powder formulation, (a) To provide a dried powder formulation prepared according to the method of claim 31, (b) Reconstituting the dried powder formulation in water or buffer solution to obtain the reconstituted dried powder formulation, Methods that include...

33. The method according to claim 32, wherein the LNP in the reconstituted dried powder formulation has a diameter of about 100 nm.

34. The method according to any one of claims 31 to 33, wherein the weight ratio of sucrose or trehalose to the total lipids in the LNP is at least about 5, at least about 11, or at least about 15.

35. The method according to any one of claims 31 to 34, wherein the concentration of sucrose or trehalose after reconstitution is greater than about 2% (w / v), greater than about 5% (w / v), or greater than about 7% (w / v).

36. The method according to any one of claims 31 to 35, wherein the concentration of sucrose or trehalose after reconstitution is about 2% (w / v) to about 10% (w / v).

37. The method according to any one of claims 32 to 36, wherein the particle size of the reconstituted dried powder formulation is less than about 120 nm.

38. The method according to any one of claims 32 to 37, wherein the particle size of the reconstructed LNP is about 80 nm to about 120 nm.

39. The method according to any one of claims 32 to 38, wherein the particle size of the reconstructed LNP is about 80 nm to about 115 nm.

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