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WO2024218166A1 - Formulations de poudre sèche reconstituables et leurs procédés d'utilisation - Google Patents

Formulations de poudre sèche reconstituables et leurs procédés d'utilisation Download PDF

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Publication number
WO2024218166A1
WO2024218166A1 PCT/EP2024/060446 EP2024060446W WO2024218166A1 WO 2024218166 A1 WO2024218166 A1 WO 2024218166A1 EP 2024060446 W EP2024060446 W EP 2024060446W WO 2024218166 A1 WO2024218166 A1 WO 2024218166A1
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Prior art keywords
dry powder
mrna
powder formulation
months
storage
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PCT/EP2024/060446
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English (en)
Inventor
Ashish Sarode
Shrirang KARVE
Priyal PATEL
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Sanofi SA
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Sanofi SA
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Priority to AU2024256347A priority Critical patent/AU2024256347A1/en
Priority to CN202480026499.5A priority patent/CN120957707A/zh
Publication of WO2024218166A1 publication Critical patent/WO2024218166A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules

Definitions

  • MRT Messenger RNA therapy
  • Dry powder formulations of mRNA can be useful and advantageous (including for inhaled delivery to the lung).
  • Reconstitutable dry powder formulations can provide additional benefits, including, for example, for improved formulations useful for other routes of administration such as for parenteral administration.
  • overcoming the challenges of developing both thermostable as well as reconstitutable dry powder formulations of mRNA-LNPs have presented challenges in the development of dry powder formulations suitable for, e.g., parenteral administration.
  • dry powder formulations that can provide certain benefits and improvements.
  • dry powder formulations provided herein can be reconstitutable and therefore suitable for a variety of applications and provide various therapeutic benefits.
  • methods of making and reconstituting dry powder formulations as well as methods of use of the dry powder formulations are also described herein.
  • the present disclosure encompasses a dry powder formulation for reconstitution comprising: (a) an excipient that is sucrose or trehalose; and (b) a lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) encapsulated by one or more lipids; and wherein the weight (w/w) ratio of the excipient of (a) and the total lipids in the LNP of (b) is at least about 5.
  • the excipient is sucrose.
  • the excipient is trehalose.
  • the w/w ratio of the excipient to the total lipids in the LNP is at least about 5.6, 11, or 15.
  • the w/w ratio of the excipient to the total lipids in the LNP is at least about 11.1 or at least about 15.6.
  • the LNP comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • the present disclosure encompasses a dry powder formulation for reconstitution comprising: (a) an excipient that is a sugar or sugar alcohol; and (b) a lipid nanoparticle (LNP) comprising messenger RNA (mRNA) encapsulated by one or more lipids; and wherein the weight (w/w) ratio of the excipient of (a) and total lipids in the LNP of (b) is at least about 5.
  • the sugar or the sugar alcohol is selected from sucrose, mannitol, xylitol, lactose, and trehalose.
  • the sugar or the sugar alcohol is trehalose.
  • the sugar or the sugar alcohol is sucrose.
  • the w/w ratio of the sugar or the sugar alcohol to the total lipids in the LNP is at least about 5.6, 11, or 15. In some embodiments, the w/w ratio of the sugar or the sugar alcohol to the total lipids in the LNP 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.
  • the particle size (e.g., mean particle size) of the reconstituted dry powder formulation is less than about 120 nm. In some embodiments, the encapsulation rate is greater than 60%. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is less than 5 pm. In some embodiments, the dry powder formulation is stable after prolonged storage, e.g., after prolonged storage at 2-8°C (e.g., at 4°C). In some embodiments, the mRNA maintains an integrity of 80% or greater after storage, e.g., storage at 2-8°C (e.g., at 4°C), for at least six months.
  • the mRNA maintains an integrity of 80% or greater, e.g., after storage at 2-8°C (e.g., 4°C), for up to 1 year. In some embodiments, the mRNA maintains an integrity of 80% or greater, e.g., after storage at 2-8°C (e.g., 4°C), for at least 1 year. In some embodiments, the mRNA maintains an integrity of about 80% or greater, e.g., after storage at 2-8°C (e.g., 4°C), for more than 1 year.
  • the mRNA encodes a therapeutic protein. In some embodiments, the mRNA encodes an antigen. In some embodiments, the formulation is suitable for a vaccine. In some embodiments, the formulation is suitable for parenteral delivery once reconstituted. In some embodiments, the formulation is suitable for intramuscular, intravenous, or subcutaneous delivery once reconstituted. In some embodiments, the formulation is suitable for mucosal delivery once reconstituted. In some embodiments, the formulation is suitable for oral, sublingual, or intranasal delivery once reconstituted.
  • the present disclosure encompasses a method of delivering mRNA in vivo, the method comprising administering to a subject in need thereof a reconstituted form of the dry powder formulation of the present disclosure.
  • the present disclosure encompasses a method of treating a disease or disorder in a subject, the method comprising administering to the subject a reconstituted form of the dry powder formulation of the present disclosure.
  • the reconstituted form of the dry powder formulation is administered subcutaneously, intravenously, or intramuscularly.
  • the present disclosure encompasses a reconstituted form of the dry powder formulation of the present disclosure for use in treating a disease or disorder in a subject.
  • the present disclosure encompasses the use of a reconstituted form of the dry powder formulation of the present disclosure in the manufacture of a medicament for treating a disease or disorder in a subject.
  • the reconstituted form of the dry powder formulation is formulated for subcutaneous, intravenous, or intramuscular administration.
  • the present disclosure encompasses a method of preparing a dry powder formulation for reconstitution, the method comprising: (a) combining a first mixture comprising lipid nanoparticles (LNPs) and an ethanolic solution, thereby obtaining a second mixture, wherein the LNPs comprise a messenger RNA (mRNA) encapsulated by one or more lipids, and wherein the ethanolic solution comprises an excipient that is sucrose or trehalose at a concentration of about 1-11% w/v, and (b) spray-drying the second mixture, thereby obtaining the dry powder formulation.
  • LNPs lipid nanoparticles
  • mRNA messenger RNA
  • the ethanolic solution comprises an excipient that is sucrose or trehalose at a concentration of about 1-11% w/v
  • the present disclosure encompasses a method of preparing a reconstituted dry powder formulation, the method comprising: (a) providing a dry powder formulation prepared according to the present disclosure; and (b) reconstituting the dry powder formulation in water or buffer, thereby obtaining the reconstituted dry powder formulation.
  • the LNPs in the reconstituted dry powder formulation have a diameter of about 100 nm.
  • 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.
  • the concentration of sucrose or trehalose is greater than 2% (w/v), greater than 5% (w/v), or greater than 7% (w/v) post-reconstitution. In some embodiments, the concentration of sucrose or trehalose is from about 2% (w/v) to about 10% (w/v) post- reconstitution.
  • the particle size of the reconstituted dry powder formulation e.g., the mean particle size of the LNP in the reconstituted formulation
  • the reconstituted LNP particle size is between 80-120 nm.
  • the reconstituted LNP particle size is between 80-115 nm.
  • FIG. 1A and FIG. IB show Dry powder product (DPP) manufactured using mRNA- LNP formulations with xylitol and lactose, respectively, as excipients did not spray dry well.
  • FIG. 2A shows the aggregates observed in the reconstituted Dry Powder (DP) with 7% mannitol (w/v) as excipient under 400x magnification (right) as compared to water alone (left), as described in Example 2.
  • FIG. 2B shows the appearances of reconstituted dry powder with 7% (left), 5% (middle), and 2.5% (right) mannitol (w/v) as excipient, as described in Example 2.
  • FIG. 3A shows no aggregates were observed in reconstituted DP with 5% sucrose (w/v) as excipient under microscope (right) as compared to water alone (left), as described in Example 2.
  • FIG. 3B shows the appearances of reconstituted dry powder with 7% (left), 5% (middle), and 2.5% (right) sucrose (w/v) as excipient, as described in Example 2.
  • FIG. 3C shows DPP manufactured using mRNA-LNP formulation with 2.5% sucrose (w/v) as excipient did not spray dry well and the liquid formulation adhered to the cyclone separator, as described in Example 2.
  • FIG. 4A shows the appearances of reconstituted dry powder with 7% (right), 5% (middle), and 2.5% (left) trehalose (w/v) as excipient, as described in Example 2.
  • FIG. 4B shows the appearance of reconstituted dry powder with 1.25% trehalose (w/v) as excipient, as described in Example 2.
  • FIG. 4C shows no aggregates were observed in reconstituted DP with 2.5% trehalose (w/v) as excipient under microscope (right) as compared to water alone (left), as described in Example 2.
  • FIG. 4A shows the appearances of reconstituted dry powder with 7% (right), 5% (middle), and 2.5% (left) trehalose (w/v) as excipient, as described in Example 2.
  • FIG. 4B shows the appearance of reconstituted dry powder with 1.25% trehalose (w/v) as excipient, as described in Example 2.
  • FIG. 4C shows no aggregates were observed in reconstituted DP with
  • 4D shows DPP manufactured using mRNA- LNP formulation with 1.25% trehalose (w/v) as excipient did not spray dry well with most of the liquid formulation adhered to the cyclone separator and only little dry powder collected in the collection vessel (middle) as compared to mRNA-LNP formulation with 7% trehalose (w/v) as excipient (right), as described in Example 2.
  • FIG. 5 shows the appearances of reconstituted dry powder with trehalose added as part of the reconstituting solvent, as described in Example 2. Concentrations shown (7% and 5% (w/v)) are the concentrations of trehalose in the post reconstitution compositions.
  • FIG. 6A-FIG. 6D are graphs showing changes of particle size (FIG. 6A), poly dispersity index (PDI; FIG. 6B), encapsulation efficiency (EE; FIG. 6C), and mRNA integrity (FIG. 6D) in post reconstitution compositions with 5% trehalose (w/v) during the course of 12 months at various temperatures (25°C, 4°C, or -20°C).
  • PDI poly dispersity index
  • EE encapsulation efficiency
  • FIG. 6D mRNA integrity
  • FIG. 7A-FIG. 7C show mRNA integrity of post reconstitution compositions with 5% trehalose (w/v) as measured by capillary electrophoresis at 25°C, 4°C and -20°C, respectively, after storage for 9 months.
  • FIG. 7D-FIG. 7F show mRNA integrity of post reconstitution compositions with 5% trehalose (w/v) as measured by capillary electrophoresis at 25°C, 4°C and -20°C, respectively, after storage for 6 months.
  • FIG. 7J shows the capillary electrophoresis plot of an mRNA standard as a control.
  • FIG. 8A is a graph showing post reconstitution of the trehalose dry powder (“Trehalose DP”) shows no change in mRNA integrity post spray drying as compared to a control mRNA (“Standard”), as described in Example 5.
  • FIG. 8B is a graph showing OTC expression in mice after intravenous injection of a liquid control and the reconstituted trehalose dry powder.
  • FIG. 9 is a graph showing human erythropoietin (hEPO) expression in mice after intramuscular injection of a liquid control and the reconstituted trehalose dry powder.
  • FIG. 10A-FIG. 10C are graphs showing no significant change in particle size, PDI, encapsulation efficiency, and mRNA integrity for three different DPPs after storage at 2-8°C over the course of 12 months, as described in Example 8.
  • FIG. 10A also shows no significant change in the hEPO expression over the course of 12 months.
  • FIG. 10A DPP containing mRNA encoding hEPO (“cKK-ElO-hEPO Dry Powder”);
  • FIG. 10B DPP containing mRNA encoding an influenza antigen (“cKK-ElO-moono-Flu Dry Powder”);
  • FIG. 10A DPP containing mRNA encoding an influenza antigen
  • FIG. 11A is a graph showing no significant change in particle size, PDI, encapsulation efficiency, and mRNA integrity for a DPP prepared with 4 different influenza mRNAs (QIV-Flu) after storage at 2-8°C over the course of 10 months, as described in Example 8.
  • FIG. 11A is a graph showing no significant change in particle size, PDI, encapsulation efficiency, and mRNA integrity for a DPP prepared with 4 different influenza mRNAs (QIV-Flu) after storage at 2-8°C over the course of 10 months, as described in Example 8.
  • FIG. 11B shows the capillary electrophoresis profiles of the QIV-Flu mRNA (“QIV mRNA”; top), the mRNA extracted from the reconstituted DPP at TO (“Dry Powder TO”; middle), and the mRNA extracted from the reconstituted DPP after storage at 2-8°C for 5.5 months (“Dry Powder 5.5 Months”; bottom).
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient’s circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).
  • circulation system e.g., serum
  • systemic distribution also systematically distributed and taken up by other tissues
  • delivery is oral delivery, intramuscular, intravenous, subcutaneous, sublingual, or buccal delivery.
  • encapsulation refers to the process of confining a nucleic acid molecule within a nanoparticle.
  • Encapsulation efficiency refers to the amount of a therapeutic and/or prophylactic, such as a ribonucleic acid molecule of the disclosure, that becomes part of a lipid nanoparticle (LNP), relative to the initial total amount of therapeutic and/or prophylactic used in the preparation of a LNP.
  • LNP lipid nanoparticle
  • the encapsulation efficiency may be given as 97%.
  • Encapsulation efficiency can be determined by, for instance, the RiboGreen assay or any method known in the art.
  • encapsulation may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.
  • expression refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post- translational modification of a polypeptide or fully assembled protein (e.g., antibody).
  • expression and “production,” and their grammatical equivalents, are used interchangeably.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life' is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • mean particle size' As used herein, “mean particle size” in the context of lipid nanoparticle compositions refers to the mean diameter of a nanoparticle composition. “Mean particle size” in the context of dry powders in the dry powder formulations refers to the mean diameter of the dry powders.
  • control subject is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • thermostability refers to the ability of a formulation (e.g., mRNA-nanoparticle or reconstituted mRNA- nanoparticle) to maintain its chemical structure and/or physical stability at elevated temperatures.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. 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 with which they were initially associated.
  • isolated agents are 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% pure.
  • a substance is “pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • liquid control refers to mRNA nanoparticle formulations directly formulated as liquid products, rather than dry powder formulations or reconstituted from dry powder formulations. Accordingly, as used herein, a liquid control formulation is distinct from a reconstituted formulation, which is adding at least one solvent to an mRNA encapsulated dry powder formulation.
  • Reconstitute As used herein, the term “reconstitute” or “reconstituted” or related words refer to a process of adding a liquid diluent to a dry ingredient to make a specific concentration of liquid formulation.
  • Reconstituted formulation refers to the dissolution of a dry powder, freeze-dried protein, spray-dried protein or spray- dried nucleic acids (e.g., mRNA) or solvent-precipitated protein in a diluent.
  • reconstituted formulation refers to a formulation prepared by dissolving or dispersing the mRNA-nanoparticle in an aqueous solution for administration.
  • mRNA-LNP composition refers to a composition or formulation comprising one or more mRNA molecules encapsulated in LNPs.
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) to which a provided composition (e.g., any described herein) may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes.
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • a composition e.g., any composition described herein
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • a composition e.g., any composition described herein
  • a subject such as a patient (e.g., a human patient) in need thereof (e.g., in need of a therapeutic and/or prophylactic treatment as described herein).
  • sugar includes 1-10 monosaccharide units, e.g., monosaccharides, disaccharides, trisaccharides, and oligosaccharides comprising 4-10 monosaccharide units.
  • Any sugar is useful in formulating the clostridial toxin pharmaceutical compositions disclosed herein, provided that a therapeutically effective amount of a clostridial toxin active ingredient, most preferably a botulinum toxin, is used using this sugar. It is assumed that it will be collected. In some embodiments, for example, in a lyophilized composition, the sugar may function as a lyoprotectant.
  • the sugar may function as an isotonic agent, for example in lyophilized or liquid formulations.
  • a monosaccharide is a cyclic type when a polyhydroxyaldehyde or polyhydroxyketone having 3 or more carbon atoms, such as aldose, dialdos, aldketose, ketose and diketose, and the parent monosaccharide has a (potential) carbonyl group, Deoxy sugars and amino sugars, and their derivatives.
  • Trioses such as glyceraldehyde and dihydroxyacetone as monosaccharides; tetroses such as erythrose, erythrulose and threose; pentoses such as arabinose, lyxose, ribose, ribulose, xylose and xylulose; allose, altrose, fructose, fucose, galactose, glucose Hexoses, such as sucrose and mannoheptulose; octoses such as octulose and 2-keto-3 -deoxy -mannooctonate; groose, idose, mannose, psicose, rhamnose, sorbose, tagatose, talose and trehalose; Nonoses such as sialose; and decourse.
  • An oligosaccharide is a compound in which at least two types of monosaccharide units are linked by a glycosidic bond. Depending on the number of units, they are called disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, nine sugars, decasaccharides, and the like. Oligosaccharides can be unbranched, branched, or cyclic.
  • Common disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, gentiobiose, cordierbiose, laminaribiose, mannobiose, melibiose, nigerose, rutinose, and xylobiose.
  • Common trisaccharides include, but are not limited to, raffinose, acarbose, maltotriose, and meletitol.
  • Other non-limiting 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 in its entirety.
  • polyalcohol is synonymous with “sugar alcohol”, “polyhydric alcohol”, and “polyol” and an alcohol group (CH 2 OH) instead of an aldehyde group (CHO).
  • sucrose alcohol a sugar alcohol
  • polyhydric alcohol a polyhydric alcohol
  • polyol an alcohol group (CH 2 OH) instead of an aldehyde group (CHO).
  • CHO aldehyde group
  • Non-limiting examples of polyols include glycol, glycerol, arabitol, erythritol, xylitol, maltitol, sorbitol (glucitol), mannitol, inositol, lactitol, galactitol (iditol), 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 its entirety
  • the present application provides among other things, dry powder formulations of mRNA-LNPs that provide unexpectedly superior properties, including thermostable dry powder products (DPP) as well as reconstitutable dry powder products (reconstitutability and thermostability).
  • DPP thermostable dry powder products
  • reconstitutable dry powder products reconstitutability and thermostability
  • Dry powder product (DPP) and dry powder (DP) can be used interchangeably with the term “dry powder formulation” as used herein.
  • dry powder formulations described herein can be suitable for use as a dry powder product without reconstitution as well as being suitable for use following reconstitution of the dry powder formulation as described herein.
  • the ability to reconstitute dry powder formulations described herein is particularly beneficial for use of these formulations in various modes of delivery, including but not limited to parenteral (intramuscular, intravenous, subcutaneous, etc.) and mucosal (oral, sublingual, intranasal, etc.) delivery.
  • parenteral intramuscular, intravenous, subcutaneous, etc.
  • mucosal oral, sublingual, intranasal, etc.
  • the disclosure features a dry powder formulation comprising a messenger RNA (mRNA) encapsulated in lipid nanoparticles (LNPs), where the lipid nanoparticles comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • the formulation comprises a sugar or sugar alcohol, wherein the weight (w/w) ratio of the sugar or sugar alcohol and the total lipids in the lipid nanoparticles is at least about 5.
  • the dry powder formulation can be reconstituted for parenteral administration by reconstitution with water or a buffer. The reconstituted form of the dry powder formulation is sometimes called “reconstituted dry powder formulation.”
  • the dry powder formulation comprises dry powders having a particle size (e.g., mean particle size) of between 18 pm or between about 1 pm and about 8 pm.
  • the disclosure features a method of preparing a dry powder formulation as described herein.
  • the method comprises providing a first mixture comprising lipid nanoparticles encapsulating an mRNA (i.e., mRNA-LNP), adding an excipient selected from a sugar or sugar alcohol (e.g., trehalose) to the mRNA- LNP at a concentration of between 2 and 10 % w/w, thereby obtaining a second mixture, and spray drying the second mixture.
  • a dry powder formulation is obtained that has a particle size of between 18 pm, e.g., the dry powders in the formulation have a mean particle size of between about 1 pm and about 8 pm.
  • the lipid nanoparticles used in the method of the disclosure comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • the dry powder formulation can be reconstituted with water or buffer for parenteral administration.
  • the dry powder comprising the excipient is stable for at least 1 year (e.g., after storage at 2-8°C for at least 1 year).
  • the present disclosure provides stable dry powder formulations containing mRNA- loaded lipid nanoparticles (mRNA-LNP) for therapeutic use.
  • the dry powder formulations of the present disclosure are thermostable and/or reconstitutable dry powder formulations.
  • 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 isomalto-oligosaccharides, reduced xylo-oligosaccharides, cellobiose, trehalose, raffinose, starch, dextran, maltodextrin, cyclodextrins, inulin, or any combination thereof.
  • the sugar alcohol is selected from erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, propylene glycol, glycerol (glycerin), threitol, galactitol, adonitol, dulcitol, pentaerythritol, or any combination thereof.
  • the sugar or sugar alcohol is selected from trehalose, mannitol, lactose, xylitol, and sucrose, as well as any combinations thereof.
  • the sugar is trehalose.
  • the sugar or sugar alcohol is mannitol.
  • the sugar is lactose.
  • the sugar or sugar alcohol is xylitol.
  • the sugar or sugar alcohol is sucrose.
  • the excipient and the excipient to total lipid content ratio in the dry powder formulation can provide a reconstitutable dry powder with optimal properties.
  • the weight ratio (w/w) of the sugar or sugar alcohol (e.g., sucrose or trehalose) to the total lipids in the lipid nanoparticle provides for optimum stability of the reconstitutable dry powder formulation.
  • the weight (w/w) ratio of the sugar or sugar alcohol to total lipids in the LNP is about 2-10, about 4-8, or about 4-6. In some embodiments, the weight (w/w) ratio of the sugar or sugar alcohol to total lipids in the LNP is about 2, about 3, about
  • the weight (w/w) ratio of the sugar or sugar alcohol to total lipids in the LNP is 2-10. In some embodiments, the w/w ratio of the sugar or sugar alcohol to total lipids in the LNP is 4-8. In some embodiments, the w/w ratio of the sugar or sugar alcohol to total lipids in the LNP is 4-6.
  • the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 2. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 3. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 4. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about
  • the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 6. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 7. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 8. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 9. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 10.
  • the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 11. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 12. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 13. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 14. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 15.
  • the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 16. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 17. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 18. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 19. In some embodiments, the ratio of w/w ratio of the sugar or sugar alcohol to the total lipids in the lipid nanoparticle is at least about 20.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.1. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.2. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.3. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.4.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.5. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.6. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.7. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.8. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 5.9.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.1. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.2. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.3. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.4. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.5.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.6. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.7. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.8. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 11.9.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.0. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.1. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.2. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.3. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.4.
  • the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.5 . In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.6. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.7. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.8. In some embodiments, the dry powder formulation comprises the w/w ratio of the excipient to the total lipids is at least 15.9.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sugar or sugar alcohol is selected from trehalose, mannitol, lactose, xylitol, and sucrose, as well as any combinations thereof.
  • a dry powder formulation (e.g., a reconstitutable dry powder formulation) comprising sugar or sugar alcohol disclosed herein has desirable properties including, but not limited to, better storage period, efficacy, thermostability, tissue absorption, and/or encapsulation efficacies. Exemplary, non-limiting beneficial features are described herein.
  • a dry powder formulation as described herein can provide beneficial effects in the storage and/or storage stability, including but not limited to the exemplary embodiments described herein.
  • a dry powder formulation as described herein can have an improved duration of storage (including, for example, at relatively elevated temperatures that do not require extreme cold storage).
  • a reconstitutable dry powder formulation as described herein is also characterized by their improved storage properties.
  • a reconstitutable dry powder formulation may be stored under refrigeration and remain stable (e.g., as demonstrated by minimal or no losses in their intended pharmaceutical or biological activity) for extended periods of time (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 values and subranges therebetween, or longer upon storage at temperatures such as those described herein, including at about, for example 2-8°C (e.g., 4°C) or -20°C).
  • stable e.g., as demonstrated by minimal or no losses in their intended pharmaceutical or biological activity
  • extended periods of time 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 values and subranges therebetween, or longer upon storage at temperatures such as those described herein, including at
  • the duration of storage of a dry powder formulation 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% (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following storage, including according to any exemplary duration, and/or temperature as described herein.
  • reconstitutable dry powder formulation described herein provides comparable duration of storage as compared to the dry powder formulation prior to reconstitution.
  • the duration of storage of a 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% (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following reconstitution, including according to any method as described herein.
  • Stability e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%
  • a reconstitutable dry powder formulation as described herein can provide beneficial effects in the stability of the formulation, including but not limited to the exemplary embodiments described herein.
  • the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein is stable for at least 1 week. In some embodiments, the reconstitutable dry powder formulation is stable for at least 2 weeks. In some embodiments, the reconstitutable dry powder formulation is stable for at least 3 weeks. In some embodiments, the reconstitutable dry powder formulation is stable for at least 4 weeks. In some embodiments, the reconstitutable dry powder formulation is stable for at least 5 weeks. In some embodiments, the reconstitutable dry powder formulation is stable for at least 1 month. In some embodiments, the reconstitutable dry powder formulation is stable for at least 2 months. In some embodiments, the reconstitutable dry powder formulation is stable for at least 3 months.
  • the stability of the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein is increased by at least about 1 week compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 2 weeks compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 3 weeks compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 4 weeks compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least about 5 weeks compared to liquid controls.
  • the stability of the reconstitutable dry powder is increased by at least 1 month compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 2 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 3 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 4 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 5 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 6 months compared to liquid controls.
  • the stability of the reconstitutable dry powder is increased by at least 7 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 8 months compared to liquid controls. In the stability of the reconstitutable dry powder formulation is increased by at least 9 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 10 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder is increased by at least 11 months compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by at least 1 year compared to liquid controls. In some embodiments, the stability of the reconstitutable dry powder formulation is increased by 1 year or more compared to liquid controls.
  • the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein comprises improved thermostability when compared to liquid controls.
  • the reconstitutable dry powder formulation is stable at 4°C for at least 1 week.
  • reconstitutable the dry powder formulation is stable at 4°C for at least 2 weeks.
  • the reconstitutable dry powder formulation is stable at 4°C for at least 3 weeks.
  • the reconstitutable dry powder formulation is stable at 4°C for at least 4 weeks.
  • the reconstitutable dry powder formulation is stable at 4°C for at least about 5 weeks.
  • the reconstitutable dry powder formulation is stable at 4°C for 1 month. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 2 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 3 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 4 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 5 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 6 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 7 months.
  • the reconstitutable dry powder formulation is stable at 4°C for at least 8 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 9 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 10 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 11 months. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for at least 1 year. In some embodiments, the reconstitutable dry powder formulation is stable at 4°C for more than 1 year.
  • the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein is stable at 25°C for at least 1 week. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 2 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 3 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 4 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least about 5 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for 1 month.
  • the reconstitutable dry powder formulation is stable at 25°C for at least 2 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 3 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 4 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 5 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 6 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 7 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 8 months.
  • the reconstitutable dry powder formulation is stable at 25°C for at least 9 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 10 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 11 months. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for at least 1 year. In some embodiments, the reconstitutable dry powder formulation is stable at 25°C for more than 1 year.
  • the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein is stable at -20°C for at least 1 week. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 2 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 3 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 4 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 5 weeks. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for 1 month.
  • the reconstitutable dry powder formulation is stable at -20°C for at least 2 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 3 months. In some embodiments, the reconstitutable dry powder formulation is stable at - 20°C for at least 4 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 5 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 6 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 7 months. In some embodiments, the reconstitutable dry powder formulation is stable at - 20°C for at least 8 months.
  • the reconstitutable dry powder formulation is stable at -20°C for at least 9 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 10 months. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for at least 11 months. In some embodiments, the reconstitutable dry powder formulation is stable at - 20°C for at least 1 year. In some embodiments, the reconstitutable dry powder formulation is stable at -20°C for more than 1 year.
  • a reconstitutable dry powder formulation described herein demonstrates a negligible reduction in pharmacological or biological activity (e.g., less than about a 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 therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide) (e.g., as compared to prior to dry powder formulation or a control formulation without spray-drying or the dry powder formulation prior to storage).
  • pharmacological or biological activity e.g., less than about a 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 therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide
  • a reconstitutable dry powder formulation described herein provides comparable stability as compared to an equivalent non-dry powder formulation.
  • the stability does not appreciably change during storage of the dry powder formulation, including any exemplary, storage, duration, and/or temperature described herein.
  • 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 therebetween (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • a reconstitutable dry powder formulation described herein provides comparable stability as compared to the dry powder formulation prior to reconstitution.
  • the stability of a reconstitutable 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following reconstitution, including according to any method as described herein.
  • mRNA integrity e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%
  • a dry powder formulation as described herein can provide beneficial effects in maintaining the integrity of the mRNA encapsulated within the LNP, including but not limited to the exemplary embodiments described herein.
  • the mRNA maintains an integrity of 60% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 64% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 66% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 68% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 72% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 76% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 78% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 80% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 84% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 88% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months orlonger, including all values and ranges therebetween. In some embodiments, the mRNA maintains an integrity of 90% or greater for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer, including all values and ranges therebetween.
  • the mRNA maintains an integrity of 60% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 64% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 66% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 68% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 72% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 76% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 78% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 80% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 84% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 88% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 60% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 64% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at 25 °C for at least six months.
  • the mRNA maintains an integrity of 66% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 68% or greater after storage at 25 °C for at least six months. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at 25 °C for at least six months.
  • the mRNA maintains an integrity of 72% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage at 25 °C for at least six months. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 76% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 78% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 80% or greater after storage at 25 °C for at least six months. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 84% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage at 25 °C for at least six months. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 88% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at 25 °C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at 25 °C for at least six months.
  • the mRNA maintains an integrity of 60% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 64% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at - 20°C for at least six months.
  • the mRNA maintains an integrity of 66% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 68% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at - 20°C for at least six months.
  • the mRNA maintains an integrity of 72% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 76% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at - 20°C for at least six months.
  • the mRNA maintains an integrity of 78% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 80% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at - 20°C for at least six months.
  • the mRNA maintains an integrity of 84% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 88% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of about 60% or greater, such as 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 therebetween, after storage at 2-8°C (e.g., about 4°C) for at least about a year.
  • 2-8°C e.g., about 4°C
  • the mRNA maintains an integrity of about 70% or greater after storage at 2-8°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 75% or greater after storage at 2-8°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 80% or greater after storage at 2- 8°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 85% or greater after storage at 2-8°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 90% or greater after storage at 2-8°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 95% or greater after storage at 2-8°C for at least about a year.
  • the mRNA maintains an integrity of about 70% or greater after storage at 4°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 75% or greater after storage at 4°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 80% or greater after storage at 4°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 85% or greater after storage at 4°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 90% or greater after storage at 4°C for at least about a year. In some embodiments, the mRNA maintains an integrity of about 95% or greater after storage at 4°C for at least about a year.
  • a reconstitutable dry powder formulation described herein provides comparable mRNA integrity as compared to an equivalent non-dry powder formulation.
  • the mRNA integrity does not appreciably change during storage of the reconstitutable dry powder formulation, including any exemplary, storage, duration, and/or temperature described herein.
  • the mRNA integrity of the reconstitutable 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • a reconstituted dry powder formulation described herein provides comparable mRNA integrity as compared to the dry powder formulation prior to reconstitution.
  • the mRNA integrity of a 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% (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following reconstitution, including according to any method as described herein.
  • Particle size e.g., Particle size
  • a reconstitutable dry powder formulation as described herein can provide beneficial effects in the particle size of the LNPs of the formulation, e.g., in maintaining the particle size (e.g., mean particle size) of the LNPs, including but not limited to the exemplary embodiments described herein.
  • the reconstitutable dry powder formulation described herein comprises dry powders having a particle size (e.g., mean particle size) of between 0.5-10 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is between 1-5 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is between 1-4 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is between 1-3 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is between 1-2 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is about 1 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 2 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 3 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 4 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 5 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 6 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the formulation is about 7 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 8 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 9 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powders in the formulation is about 10 pm.
  • the particle size (e.g., mean particle size) of the dry powders in the reconstitutable dry powder formulation disclosed herein is desirable for delivery into various tissues.
  • dry powder formulations comprising particle size (e.g., mean particle size) of about 5 microns or less are especially useful for pulmonary delivery.
  • dry powder formulations comprising trehalose have been found to have a particle size of 5 microns or less and may be suited to pulmonary delivery.
  • a reconstitutable dry powder formulation described herein provides comparable particle size (e.g., mean particle size) as compared to an equivalent non-dry powder formulation.
  • the particle size e.g., mean particle size
  • the particle size does not appreciably change during storage of a dry powder formulation, including any exemplary storage, duration, and/or temperature described herein.
  • the particle size (e.g., mean particle size) of a reconstitutable 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • reconstitutable dry powder formulation described herein provides comparable particle size (e.g., mean particle size) as compared to the dry powder formulation prior to reconstitution.
  • the particle size (e.g., mean particle size) of a reconstitutable 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following reconstitution, including according to any method as described herein. e. Delivery Routes
  • reconstitutable dry powder formulations comprising sugar or sugar alcohol as described herein enable administration of the mRNA-nanoparticle as a liquid formulation at a desirable dosage.
  • the reconstituable dry powder formulation with dry powders having 6 microns or less, 5 microns or less, 4 microns or less, 3 microns or less, 2 microns or less, 1 micron or less, including all values and ranges therebetween, may be suitable for pulmonary delivery.
  • the pharmaceutical compositions comprising the dry powder formulations for reconstitution as described herein may be administered to a subject in need thereof subconjunctival, sublingual by any one of several routes that effectively delivers an effective amount of the compound.
  • Non-limiting examples of suitable administrative routes include topical, oral, nasal, intrathecal, enteral, buccal, sublingual, transdermal, rectal, vaginal, intraocular, and parenteral administration, including subcutaneous, intravenous, intramuscular, intrasternal, intracavemous, intrameatal, and intraurethral injection and/or infusion.
  • the reconstitutable dry powder formulation is suitable for mucosal delivery. In some embodiments, the reconstitutable dry powder formulation is suitable for oral delivery. In some embodiments, the reconstitutable dry powder formulation is suitable for sublingual delivery. In some embodiments, the reconstitutable dry powder formulation is suitable for or intranasal delivery. In some embodiments, the reconstitutable dry powder formulation is suitable for buccal delivery. f. Improved delivery efficacy
  • a reconstituable dry powder formulation as described herein can provide beneficial effects in the delivery of the formulation to the target tissues, including but not limited to the exemplary embodiments described herein.
  • reconstituable dry powder formulations described in this disclosure are useful for improved therapeutic efficacy, including improved delivery of mRNA to the cells.
  • the reconstituable dry powder formulations have improved delivery of mRNA to various tissues compared to liquid controls.
  • the formulations described in this disclosure have improved delivery when used orally, mucosally, intravenously, intramuscularly, subcutaneously, transdermally or buccally.
  • the formulations described in this disclosure have improved delivery when used in delivery routes that are suitable for the present disclosure.
  • a reconstitutable dry powder formulation described herein provides comparable delivery efficacy as compared to an equivalent non-dry powder formulation.
  • the delivery efficacy does not appreciably change during storage of a reconstituable dry powder formulation, including any exemplary storage, duration, and/or temperature described herein.
  • the delivery efficacy of a 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • reconstitutable dry powder formulation described herein provides comparable delivery efficacy as compared to the dry powder formulation prior to reconstitution.
  • the delivery efficacy of a reconstituable 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., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween, following reconstitution, including according to any method as described herein.
  • Sugar -lipid ratios e.g., sugar -lipid ratios
  • Reconstitutable dry powder formulations described herein can have an optimal particle size for tissue specific delivery.
  • the reconstitutable dry powder formulation comprising trehalose is used for pulmonary delivery.
  • the ratio of concentration of trehalose to total lipid concentration is greater than 1. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 2. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 3. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 4. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 5. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 6. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 7.
  • the ratio of concentration of trehalose to total lipid concentration is greater than 8. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 9. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 10. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 11. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 12. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 13. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 14. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 15.
  • the ratio of concentration of trehalose to total lipid concentration is greater than 16. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 17. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 18. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 19. In some embodiments, the ratio of concentration of trehalose to total lipid concentration is greater than 20. h. Encapsulation efficiencies
  • a reconstitutable dry powder formulation as described herein can provide beneficial effects in the encapsulation efficiencies of the LNPs of the formulation, including but not limited to the exemplary embodiments described herein.
  • the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein comprises higher encapsulation efficiency compared to liquid controls or dry powders without sugar or sugar alcohol.
  • the encapsulation efficiency is greater than 60%. In some embodiments, the encapsulation efficiency is greater than 61%. In some embodiments, the encapsulation efficiency is greater than 62%. In some embodiments, the encapsulation efficiency is greater than 63%. In some embodiments, the encapsulation efficiency is greater than 64%. In some embodiments, the encapsulation efficiency is greater than 65%. In some embodiments, the encapsulation efficiency is greater than 66%. In some embodiments, the encapsulation efficiency is greater than 67%.
  • the encapsulation efficiency is greater than 68%. In some embodiments, the encapsulation efficiency is greater than 69%. In some embodiments, the encapsulation efficiency is greater than 70%. In some embodiments, the encapsulation efficiency is greater than 71%. In some embodiments, the encapsulation efficiency is greater than 72%. In some embodiments, the encapsulation efficiency is greater than 73%. In some embodiments, the encapsulation efficiency is greater than 74%. In some embodiments, the encapsulation efficiency is greater than 75%. In some embodiments, the encapsulation efficiency is greater than 76%. In some embodiments, the encapsulation efficiency is greater than 77%. In some embodiments, the encapsulation efficiency is greater than 78%.
  • the encapsulation efficiency is greater than 79%. In some embodiments, the encapsulation efficiency is greater than 80%. In some embodiments, the encapsulation efficiency is greater than 81%. In some embodiments, the encapsulation efficiency is greater than 82%. In some embodiments, the encapsulation efficiency is greater than 83%. In some embodiments, the encapsulation efficiency is greater than 84%. In some embodiments, the encapsulation efficiency is greater than 85%. In some embodiments, the encapsulation efficiency is greater than 86%. In some embodiments, the encapsulation efficiency is greater than 87%. In some embodiments, the encapsulation efficiency is greater than 88%. In some embodiments, the encapsulation efficiency is greater than 89%.
  • the encapsulation efficiency is greater than 90%. In some embodiments, the encapsulation efficiency is greater than 91%. In some embodiments, the encapsulation efficiency is greater than 92%. In some embodiments, the encapsulation efficiency is greater than 93%. In some embodiments, the encapsulation efficiency is greater than 94%. In some embodiments, the encapsulation efficiency is greater than 95%. In some embodiments, the encapsulation efficiency is greater than 96%. In some embodiments, the encapsulation efficiency is greater than 97%. In some embodiments, the encapsulation efficiency is greater than 98%. In some embodiments, the encapsulation efficiency is greater than 99%.
  • the particle size (e.g., mean particle size) of the dry powders comprised in the reconstitutable dry powder formulation comprising sugar or sugar alcohol as described herein is less than 3pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 4pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 5 pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 6pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 7pm.
  • the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 8pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 9pm. In some embodiments, the particle size (e.g., mean particle size) of the reconstitutable dry powder is less than 10pm.
  • a reconstitutable dry powder formulation described herein exhibits an enhanced (e.g., increased) ability to transfect one or more target cells.
  • methods of transfecting one or more target cells generally comprise the step of contacting the one or more target cells with, for example, a dry powder formulation as described herein such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides or mRNA).
  • a dry powder formulation can be beneficial for needle-free (i.e., non-invasive) routes of administration such as, but not limited to, delivery via mucosal surfaces.
  • Reconstitutable dry powder formulations as described herein provide beneficial properties following reconstitution to the desired mRNA-LNP formulation, including but not limited to the exemplary beneficial features described herein.
  • a reconstitutable dry powder formulation as described herein can maintain desirable features following reconstitution including, but not limited to beneficial stability (e.g., as determined, for example, with reference to the particle size (e.g., mean particle size) of the reconstituted lipid nanoparticles comprising such composition).
  • beneficial stability e.g., as determined, for example, with reference to the particle size (e.g., mean particle size) of the reconstituted lipid nanoparticles comprising such composition.
  • reconstitution of a dry powder formulation described herein does not appreciably change or alter the particle size (e.g., mean particle size) and/or encapsulation efficiency of the lipid nanoparticles following reconstitution.
  • reconstitution of a dry powder formulation described herein does not appreciably change or alter the therapeutic efficacy of the mRNA (e.g., the integrity and/or activity of the mRNA) following reconstitution. a. Lack of aggregation
  • a reconstituted dry powder formulation as described herein can provide beneficial effects in the stability of the formulation, by for example, minimizing aggregation, including but not limited to the exemplary embodiments described herein.
  • disclosed herein are dry powder compositions wherein upon reconstitution (e.g., according to methods described herein) the lipid nanoparticles do not flocculate or aggregate, or alternatively demonstrated limited or negligible flocculation or aggregation (e.g., a determined by the particle size (e.g., mean particle size) of the reconstituted lipid nanoparticles).
  • the lipid nanoparticles upon reconstitution of a dry powder formulation as described herein, have a geometric particle size distribution Dv50 of less than about 500nm (e.g., less than about 300 nm, 200nm, 150nm, 125nm, 120nm, lOOnm, 75nm, 50nm, 25nm, or smaller, including all values and ranges therebetween).
  • Dv50 geometric particle size distribution
  • the lipid nanoparticles upon reconstitution of a dry powder formulation as described herein, have a geometric particle size distribution Dv90 of less than about 750nm (e.g., less than about 700nm, 500nm, 300nm, 200nm, 150nm, 125nm, lOOnm, 75nm, 50nm, 25nm, or smaller, including all values and ranges therebetween).
  • the reconstituted dry powder formulation upon reconstitution with an appropriate rehydration media, demonstrates pharmacological or biological activity comparable with that observed prior to dry powder formulation.
  • the pharmacological or biological activity of an encapsulated polynucleotide in a reconstituted formulation is equivalent to that observed prior to dry powder formulation of the composition, or alternatively demonstrates a negligible reduction in pharmacological or biological activity (e.g., less than about a 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 therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide).
  • a reconstituted composition e.g., lipid nanoparticles
  • Such methods generally comprise the step of contacting the one or more target cells with a reconstituted dry powder formulation as described herein such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides or mRNA).
  • the reconstitutable dry powder product as disclosed herein comprising mRNA-nanoparticle and excipients is reconstituted with a solvent.
  • the reconstitution of the dry powder product provides advantages for parenteral administration.
  • the solvent is water.
  • the solvent is a buffer.
  • the buffers or pH-adjusting agent in emulsion compositions is used to adjust the pH to a desirable range.
  • Exemplary buffers include but are not limited to phosphate buffer, citrate buffer, tris buffer, carbonate buffer, succinate buffer, maleate buffer and borate buffer.
  • the buffer is selected from the group of phosphate buffered saline (PBS), modified PBS and citrate buffer.
  • PBS phosphate buffered saline
  • the buffer can be any buffer that is suitable for the formulations and methods described in the present disclosure.
  • the reconstitution of the dry powder product involves, reconstitution in a buffer directly.
  • the reconstitution of the dry powder product in a buffer comprises the steps: (1) reconstitution with water and (2) reconstitution with a buffer.
  • a reconstituted dry powder formulation as described herein can provide beneficial effects in the stability of the formulation, including but not limited to the exemplary embodiments described herein.
  • the reconstituted dry powder formulation comprising sugar or sugar alcohol as described herein is stable for at least 1 week. In some embodiments, the reconstituted dry powder formulation is stable for at least 2 weeks. In some embodiments, the reconstituted dry powder formulation is stable for at least 3 weeks. In some embodiments, the reconstituted dry powder formulation is stable for at least 4 weeks. In some embodiments, the reconstituted dry powder formulation is stable for at least 1 month. In some embodiments, the reconstituted dry powder formulation is stable for at least 2 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 3 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 4 months.
  • the reconstituted dry powder formulation is stable for at least 5 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 6 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 7 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 8 months. In some embodiments, the reconstituted dry powder formulation is stable for at least 9 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.
  • the stability of the reconstituted dry powder formulation comprising sugar or sugar alcohol is increased by at least 1 week compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 2 weeks compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 3 weeks compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 4 weeks compared to liquid controls. In some embodiments, the stability of the dry powder is increased by at least 1 month compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 2 months compared to liquid controls.
  • the stability of the reconstituted dry powder formulation is increased by at least 3 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 4 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 5 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 6 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 7 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 8 months compared to liquid controls. In the stability of the reconstituted dry powder formulation is increased by at least 9 months compared to liquid controls.
  • the stability of the reconstituted dry powder formulation is increased by at least 10 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 11 months compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by at least 1 year compared to liquid controls. In some embodiments, the stability of the reconstituted dry powder formulation is increased by 1 year or more compared to liquid controls. [121] In some embodiments, the reconstituted dry powder formulation comprising sugar or sugar alcohol comprises better thermostability when compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 1 week compared to liquid controls.
  • the reconstituted dry powder formulation is stable at 4°C for at least 2 weeks. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 3 weeks compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 4 weeks compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for 1 month compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 2 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 3 months compared to liquid controls.
  • the reconstituted dry powder formulation is thermostable at 4°C for at least 4 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 5 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 6 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 7 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 8 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 9 months compared to liquid controls.
  • the reconstituted dry powder formulation is thermostable at 4°C for at least 10 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 11 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for at least 1 year compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is thermostable at 4°C for more than 1 year compared to liquid controls.
  • the reconstituted dry powder formulation is stable at 25°C for at least 3 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 4 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 5 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 6 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 7 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at 25°C for at least 8 months compared to liquid controls.
  • the reconstituted dry powder formulation comprising sugar or sugar alcohol is stable at -20°C for at least 1 week compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 2 weeks compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 3 weeks compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 4 weeks compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for 1 month compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 2 months compared to liquid controls.
  • the reconstituted dry powder formulation is stable at -20°C for at least 9 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 10 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 11 months compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for at least 1 year compared to liquid controls. In some embodiments, the reconstituted dry powder formulation is stable at -20°C for more than 1 year compared to liquid controls.
  • the reconstituted dry powder formulation demonstrates a negligible reduction in pharmacological or biological activity (e.g., less than about a 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 therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide) (e.g., as compared to prior to dry powder formulation).
  • pharmacological or biological activity e.g., less than about a 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 therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide
  • the reconstituted dry powder formulation described herein provides comparable stability as compared to an equivalent non-dry powder formulation.
  • the stability does not appreciably change during storage of a dry powder formulation, including any exemplary storage, duration, and/or temperature described herein.
  • the stability of a 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%, including all values and ranges therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • the reconstituted dry powder formulation described herein provides comparable stability as compared to the reconstituted dry powder formulation prior to reconstitution.
  • the stability of a 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%, 50%, including all values and ranges therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following reconstitution, including according to any method as described herein.
  • Storage e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween
  • a reconstituted dry powder formulation as described herein can provide beneficial effects in the storage and/or storage stability, including but not limited to the exemplary embodiments described herein.
  • a dry powder formulation as described herein can have an improved duration of storage (including, for example, at relatively elevated temperatures that do not require extreme cold storage).
  • a reconstituted dry powder formulation as described herein is also characterized by their improved storage properties.
  • a reconstituted dry powder formulation may be stored under refrigeration and remain stable (e.g., as demonstrated by minimal or no losses in their intended pharmaceutical or biological activity) for extended periods of time (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 upon storage at temperatures such as those described herein, including at about 4°C or -20°C).
  • the duration of storage 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%, 50%, including values and ranges therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • the reconstituted dry powder formulation described herein provides comparable duration of storage as compared to the dry powder formulation prior to reconstitution.
  • the duration of storage of a 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%, 50%, including ranges and values therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following reconstitution, including according to any method as described herein. e. Particle size
  • a reconstituted dry powder formulation as described herein can provide beneficial effects in the particle size (e.g., mean particle size) of the LNPs of the formulation, including but not limited to the exemplary embodiments described herein.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 80 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 81 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 82 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 83 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 84 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 85 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 86 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 87 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 88 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 89 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 90 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 91 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 92 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 93 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 94 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 95 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 96 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 97 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 98 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 99 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 105 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 106 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 107 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 108 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 109 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 110 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 11 Inm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 112 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 113 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 114 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 115 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 116 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 117 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 118 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 119 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 125 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 126 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 127 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 128 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 129 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 130 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 131 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 132 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 133 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 134 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 140 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 141 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 142 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 143 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 144 nm.
  • the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 145 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 146 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 147 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 148 nm. In some embodiments, the particle size of the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 149 nm.
  • the mean particle size of the LNPs in the reconstituted dry powder formulation comprising sugar or sugar alcohol as excipients is less than 150 nm, such as 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, 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 therebetween.
  • a reconstituted dry powder formulation described herein provides comparable particle size (e.g., mean particle size of the LNPs in the reconstituted formulation) as compared to an equivalent non-dry powder formulation.
  • the particle size e.g., mean particle size of the reconstituted LNPs
  • the particle size does not appreciably change during storage of the reconstituted dry powder formulation, including any exemplary storage, duration, and/or temperature described herein.
  • the particle size of the reconstituted dry powder formulation (e.g., mean particle size of the 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 therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following storage, including according to any exemplary storage, duration, and/or temperature as described herein.
  • the reconstituted dry powder formulation described herein provides comparable particle size (e.g., mean particle size of the LNPs in the reconstituted formulation) as compared to the reconstituted dry powder formulation prior to reconstitution.
  • the particle size of a reconstituted dry powder formulation e.g., mean particle size of the LNPs in the reconstituted formulation
  • the reconstituted dry powder formulation as described herein can provide beneficial effects in the integrity of the mRNA cargo, including but not limited to the exemplary embodiments described herein.
  • the mRNA maintains an integrity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, 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.
  • the mRNA maintains an integrity of 60% or greater 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.
  • the mRNA maintains an integrity of 61% or greater 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, the mRNA maintains an integrity of 62% or greater after storage for
  • the mRNA maintains an integrity of 63% or greater 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, the mRNA maintains an integrity of 64% or greater 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.
  • the mRNA maintains an integrity of 65% or greater 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, the mRNA maintains an integrity of 66% or greater 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, the mRNA maintains an integrity of 67% or greater after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the mRNA maintains an integrity of 68% or greater 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, the mRNA maintains an integrity of 69% or greater 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.
  • the mRNA maintains an integrity of 70% or greater 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, 36months or longer. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage for
  • the mRNA maintains an integrity of 72% or greater after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the mRNA maintains an integrity of 75% or greater 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, the mRNA maintains an integrity of 76% or greater after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the mRNA maintains an integrity of 77% or greater 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, the mRNA maintains an integrity of 78% or greater 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, the mRNA maintains an integrity of 79% or greater after storage for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • the mRNA maintains an integrity of 80% or greater 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, the mRNA maintains an integrity of 81% or greater 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.
  • the mRNA maintains an integrity of 82% or greater 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, the mRNA maintains an integrity of 83% or greater 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.
  • the mRNA maintains an integrity of 84% or greater 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, the mRNA maintains an integrity of 85% or greater 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.
  • the mRNA maintains an integrity of 86% or greater 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, the mRNA maintains an integrity of 87% or greater 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.
  • the mRNA maintains an integrity of 88% or greater 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, the mRNA maintains an integrity of 89% or greater 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, the mRNA maintains an integrity of 90% or greater for 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36 months or longer.
  • the mRNA maintains an integrity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage 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, 36 months.
  • the mRNA maintains an integrity of 60% or greater after storage at 4 °C for at least six months.
  • the mRNA maintains an integrity of 61% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 62% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 64% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 66% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 68% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 72% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 74% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 76% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 78% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 80% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 84% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 86% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 88% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 91% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 92% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 93% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 94% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 95% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 96% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 97% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 98% or greater after storage at 4°C for at least six months. In some embodiments, the mRNA maintains an integrity of 99% or greater after storage at 4°C for at least six months.
  • the mRNA maintains an integrity of 60% or greater, such as 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at, for example, 2-8°C (e.g., 4°C) for at least one year.
  • the mRNA maintains an integrity of 60% or greater, such as 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at, for example, 2-8°C (e.g., 4°C) for more than one year.
  • the mRNA maintains an integrity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 60% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 63% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 64% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 66% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 68% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 69% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 70% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 72% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 75% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 76% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 78% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 80% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 81% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 82% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 84% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 87% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 88% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 91% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 92% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 93% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of 94% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 95% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 96% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 97% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 98% or greater after storage at 25°C for at least six months. In some embodiments, the mRNA maintains an integrity of 99% or greater after storage at 25°C for at least six months.
  • the mRNA maintains an integrity of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 60% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 61% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 62% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 63% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 64% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 65% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 66% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 67% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 68% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 69% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 70% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 71% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 72% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 73% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 74% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 75% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 76% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 77% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 78% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 79% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 80% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 81% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 82% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 83% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 84% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 85% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 86% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 87% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 88% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 89% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 90% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 91% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 92% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 93% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 94% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 95% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 96% or greater after storage at -20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 97% or greater after storage at - 20°C for at least six months. In some embodiments, the mRNA maintains an integrity of 98% or greater after storage at -20°C for at least six months.
  • the mRNA maintains an integrity of 99% or greater after storage at -20°C for at least six months. [142] In some embodiments, the mRNA maintains an integrity of 60% or greater, such as 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at -20°C for at least one year. g. Delivery Routes
  • the reconstituted dry powder product comprising sugar or sugar alcohol enables administration of the mRNA-nanoparticle as a liquid formulation at a desirable dosage.
  • the term “desirable dosage” is a dose that is suitable for the reconstitutable dry powder formulation described in the present disclosure.
  • the pharmaceutical compositions comprising the reconstituted dry powder product as described herein may be administered to a subject in need thereof subconjunctival, sublingual by any one of several routes that effectively delivers an effective amount of the compound.
  • Nonlimiting examples of suitable administrative routes include topical, oral, nasal, intrathecal, enteral, buccal, sublingual, transdermal, rectal, vaginal, intraocular, and parenteral administration, including subcutaneous, intravenous, intramuscular, intrasternal, intracavemous, intrameatal, and intraurethral injection and/or infusion.
  • the reconstituted dry powder product is suitable for mucosal delivery. In some embodiments, the reconstituted dry powder product is suitable for oral delivery. In some embodiments, the reconstituted dry powder product is suitable for sublingual delivery. In some embodiments, the formulation is suitable for or intranasal delivery. In some embodiments, the reconstituted dry powder product is suitable for buccal delivery. In some embodiments, the reconstituted dry powder product is suitable for any delivery route that is suitable for the present disclosure.
  • the preparation of LNPs entails encapsulating mRNA lipids are added to the aqueous buffer containing mRNA at a certain Nitrogen (lipid) to Phosphate (nucleic acid) ratio (N/P ratio).
  • mRNA and lipids are combined with pump systems which maintain the lipid/mRNA (N/P) ratio constant throughout the process and which can also afford facile scale-up.
  • the one or more LNPs encapsulating mRNA (also referred to as mRNA-loaded LNPs or mRNA-LNPs) have a lipid:mRNA (N/P) ratio ranging from 1 to 20, 1 to 15, 1 to 10, 2 to 8, 2 to 6, or 2 to 4.
  • the one or more mRNA- loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 20.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 18.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 16. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 14. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 12. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 10.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 8. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 1 to 6. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 20. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 16.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 12. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 8. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 6. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 2 to 4.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 4 to 20. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 4 to 16. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 4 to 14. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 4 to 12.
  • the one or more mRNA-loaded lipid nanoparticles have a lipid:mRNA (N/P) ratio ranging from 4 to 10. In some embodiments, the one or more mRNA-loaded LNPs have a lipid:mRNA (N/P) ratio of 2 or 4. In some embodiments, the one or more mRNA-loaded LNPs have a lipid:mRNA (N/P) ratio of 2. In some embodiments, the one or more mRNA- loaded LNPs have a lipid:mRNA (N/P) ratio of 4.
  • the lipid nanoparticles encapsulating the mRNA have an N/P ratio of between 2 and 6. In some embodiments, the N/P ratio is between 3 and 4. In some embodiments, the N/P ratio is 3. [149] In some embodiments, a dry powder formulation comprises lipid nanoparticles that are added to the mRNA at an N:P ratio of about 1. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 2. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 3.
  • a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 4. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 5. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 6. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 7. In some embodiments, a dry powder formulation comprises lipid nanoparticles are added to the mRNA at an N:P ratio of about 8.
  • dry powder formulations described herein can have unexpectedly improved encapsulation efficiencies, including according to the further exemplary embodiments provided below.
  • the one or more mRNA-loaded lipid nanoparticles of the dry powder formulations described herein have an encapsulation efficiency of about 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween.
  • the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 70% or greater.
  • the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 71% or greater.
  • the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 72% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 73% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 74% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 75% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 76% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 77% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 78% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 79% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 80% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 81% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 82% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 83% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 84% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 85% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 86% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 87% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 88% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 89% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 90% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 91% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 92% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 93% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 94% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 95% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 96% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 97% or greater.
  • the one or more mRNA- loaded lipid nanoparticles have an encapsulation efficiency of 98% or greater. In some embodiments, the one or more mRNA-loaded lipid nanoparticles have an encapsulation efficiency of 99% or greater.
  • the reconstituted dry powder formulation described herein provides comparable encapsulation efficiencies as compared to an equivalent non-dry powder formulation.
  • the encapsulation efficiency of the one or more mRNA-loaded lipid nanoparticles of the dry powder formulations described herein does not appreciably change during storage of the reconstituted dry powder formulation, including any exemplary storage condition, duration, and/or temperature described herein.
  • the encapsulation efficiency of the one or more mRNA-loaded lipid nanoparticles 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%, 50%, including all values and ranges therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following storage, including according to any exemplary storage condition, duration, and/or temperature as described herein.
  • the encapsulation efficiency of the one or more mRNA-loaded lipid nanoparticles of the reconstituted dry powder formulation 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%, 50%, including all values and ranges therebetween, (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%, including all values and ranges therebetween) following reconstitution, including according to any method as described herein.
  • dry powder formulations described herein can be formulated to provide the optimal lipid nanoparticle size for various uses, including according to the further exemplary embodiments provided below.
  • Suitable mRNA loaded lipid nanoparticles may be made in various sizes.
  • the size of an mRNA loaded lipid nanoparticles pre-spray drying is determined by the length of the largest diameter of the lipid nanoparticle.
  • an mRNA loaded lipid nanoparticle has a size pre-spray drying no greater than about 250 nm (e.g., no greater than 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, including all values and ranges therebetween).
  • a suitable liposome has a size ranging from about 10 nm to about 250 nm (e.g., ranging from 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).
  • an mRNA loaded lipid nanoparticle has a size pre-spray drying ranging from about 100 nm to about 250 nm (e.g., ranging from about 100 nm to about 225 nm, about 100 nm to about 200 nm, about 100 nm to about 175 nm, about 100 nm to about 150 nm).
  • an mRNA loaded lipid nanoparticle has a size pre-spray drying ranging from about 10 nm to about 100 nm (e.g., ranging from 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 a particular embodiment, an mRNA loaded lipid nanoparticle has a size pre-spray drying less than about 100 nm.
  • the size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • QELS quasi-electric light scattering
  • Spray drying is a commonly used, economical, and established technique to manufacture the dry powder product for various modalities such as small molecules, peptides, and proteins.
  • the technique is continuous, scalable, suitable for heat sensitive material, can produce consistent dry powder product, and can be automated.
  • Various sugars such as lactose and mannitol are commonly used as carrier excipients to facilitate the spray drying process.
  • beneficial properties of mannitol as an excipient for spray drying of mRNA formulations include (i) altering effect on the viscoelastic properties associated to the phlegm (ii) increasing water content driven by osmotic gradient (iii) less hygroscopic compared to some other sugars like lactose (iv) non-reducing sugar with absence of aldehyde group etc.
  • amino acids such as leucine, isoleucine, and trileucine have been utilized to increase the dispersibility and decrease the MMAD of dry powder product.
  • the process involves, in general , removal of moisture from a compositi on by passing a liquid form of the composition through an apparatus.
  • a liquid formulation comprising the composition of interest is passed through a narrow inlet spray ‘atomizer’ nozzle into a first chamber, which is the drying chamber.
  • the liquid formulation is passed in a steady stream.
  • the liquid formulation is sprayed as tiny droplets into the drying chamber.
  • a stream of heated air or gas is also led into the drying chamber to form an air current. Passage of the formulation through this heated current disperses the incoming droplets, dries them into the solid particulate form.
  • This product is led into a second chamber by flow through a connector or pipe.
  • the second chamber is the cyclone powder collector.
  • the air circulation generates a cyclone, and the powder particles are collected via a vortex stream into a collection vessel attached to the outlet end.
  • the cyclone chamber is attached to an exhaust fan, which helps cool the components.
  • the inlet and outlet temperatures are operator adjustable.
  • the respective inlet and outlet temperatures, the chamber temperatures, the liquid feed fl ow rate (aspirator %), pressure, nature of the heated air current and most importantly, the compositi on of the li qui d feed are suitably adjusted for optimal drying of any particulate matter.
  • the inlet temperature is adjustable within a range of 40°C to 200°C. In some embodiments, the outlet temperature ranges between 20-70°C.
  • the relative pressure of the pump and the aspirator is also operator adjustable. 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 inter temperature was adjusted to 61 °C. In some embodiments, the inter temperature was adjusted to 62°C. In some embodiments, the inter temperature was adjusted to 63°C. In some embodiments, the inter temperature was adjusted to 64°C. In some embodiments, the inter temperature was adjusted to 65°C. In some embodiments, the inter temperature was adjusted to 66°C. In some embodiments, the inter temperature was adjusted to 67°C. In some embodiments, the inter temperature was adjusted to 68°C. In some embodiments, the inter temperature was adjusted to 69°C. In some embodiments, the inter temperature was adjusted to 70°C.
  • the inlet temperature is adjusted between 70 °C and 200 °C. In some embodiments, the inlet temperature is adjusted between 80 °C and 200 °C. In some embodiments, the inlet temperature is adjusted between 90 °C and 200 °C. In some embodiments, the inlet temperature is adjusted between 95 °C and 180 °C. In some embodiments, the inlet temperature is adjusted between 95 °C and 160 °C. In some embodiments, the inlet temperature is adjusted between 90 °C and 150 °C. In some embodiments, the inlet temperature is adjusted between 90 °C and 120 °C.
  • the inlet temperature is adjusted between 90 °C and 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 values and subranges therebetween.
  • the outlet temperature ranges between 20°C to 70°C. In some embodiments, the outlet temperature is between 30 °C to 60 °C. In some embodiments, the outlet temperature is between 20 °C to 50 °C. In some embodiments, the outlet temperature is between 30 °C to 50 °C. In some embodiments, the outlet temperature is between 40 °C to 50 °C. In some embodiments, the outlet temperature is between 45 °C and 50 °C.
  • Spray drying can be carried out using any suitable spray-drying device.
  • any suitable spray-drying device As is known to a person of ordinary skill in the art, a variety of spray-drying instruments are commercially available and can be used to practice the present disclosure. Exemplary commercially available devices suitable for the present disclosure include, but are not limited to the following: Mini Spray Dryer B-290; Nano Spray Dryer B-90 (manufactured by Buchi); Anhydro MicraSpray Dryer GMP; Anhydro MicraSpray Dryer Aseptic series (manufactured by SPX FLOW); MDL-50 and MDL-015 (manufactured by Fujisaki Electric); Versatile Mini Sprayer Dryer GAS410 (manufactured by Yamato Scientific America); LSD-1500 Mini spray dryer, MSD-8 Multi -functional laboratory spray dryer; PSD-12 Precision pharmacy spray dryer; (manufactured by Changzhou Xiandao Drying Equipment Co.
  • TALL FORM DRYERTM Multi-Stage Dryer; COMPACT DRYERTM; FILTERMATTM Spray Dryer; VERSATILE-SDTM; Fluidized Spray Dryer; MOBILE MINORTM; SDMICROTM; PRODUCTION MINORTM (manufactured by GEA Process Engineering) and many others. Convenient scale up from laboratory scale to industrial manufacturing scale is also available from several of these manufacturers.
  • Dry powders prepared according to the present disclosure contain a plurality of spray-dried particles. Residual moisture content, aerosol performance and physio-chemical stability are important parameters for spray-dried pharmaceutical products. It is determined by the sample weight loss after heating and drying, using the equation:
  • Moisture content wherein, SWb is the sample weight before heating and SW a is the sample weight after heating.
  • Perkin Elmer TGA 7 (Perkin Elmer) is an example of commercially used instrument with associated software for the measurement of residual moisture in a nanoparticle.
  • the particles of the dry powder formulation suitable for pulmonary administration comprise trehalose. In some embodiments, the particles of the dry powder formulation suitable for pulmonary administration, comprising trehalose are 5 micrometer or less.
  • spraying lipid nanoparticles using hydroalcoholic solution aids in reducing the particle size of the DPP. In some embodiments, spraying lipid nanoparticles using hydroalcoholic solution comprising sugar or sugar alcohol aids in reducing particle of DPP.
  • the sugar is mannitol, xylitol, lactose, sucrose, or trehalose.
  • 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.
  • the particle size (e.g., mean particle size) of the dry powder is between 0.5-10pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder formulation is between 1-8 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder formulation is between 1-7 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder formulation is between 1-6 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is between 1-5 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is between l-4pm.
  • the particle size (e.g., mean particle size) of the dry powder is between 1-3 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is between l-2pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 1 m. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 2pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 3 pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 4pm.
  • the particle size (e.g., mean particle size) of the dry powder is about 5pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 6pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 7pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 8pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 9pm. In some embodiments, the particle size (e.g., mean particle size) of the dry powder is about 10pm.
  • Primary particle size (e.g., mean particle size) distributions of spray-dried particles are measured by dynamic light scattering, which is expressed in terms of Z-average.
  • the Z- average is the mean, also known as the cumulant size, calculated from the intensity- weighted distribution of particle diameter and is given by the formula,
  • Poly dispersity index (PDI) is the measure of the distribution of molecular mass of a given particulate sample.
  • the poly dispersity index of the glycerol and propylene glycol based LNP is less than 0.2. In some embodiments, the poly dispersity index of the glycerol and propylene glycol based LNP is about 0.1. In some embodiments, the polydispersity index of the glycerol and propylene glycol based LNP is less than about 0.1.
  • the mRNA constitutes greater than about 2% of the dry powder formulation by weight. In some embodiments, the mRNA constitutes greater than about 3% of the dry powder formulation by weight. In some embodiments, the mRNA constitutes greater than about 4% of the dry powder formulation by weight. [171] It was observed that the dry powder formulation of mRNA of the present disclosure had high stability. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 80% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 81% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 82% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 83% integrity.
  • the mRNA in the dry powder formulation maintains greater than about 84% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 85% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 86% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 87% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 88% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 89% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 90% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 91% integrity.
  • the mRNA in the dry powder formulation maintains greater than about 92% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 93% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 94% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 95% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 96% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 97% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 98% integrity. In some embodiments, the mRNA in the dry powder formulation maintains greater than about 99% integrity.
  • the integrity of the mRNA be maintained after multiple freeze-thaw cycles and/or for prolonged periods of time for maintaining therapeutic benefit.
  • the mRNA maintains integrity of 80% or greater after storage at room temperature for 6 months or longer. In some embodiments, the mRNA maintains integrity of 80% or greater after storage at room temperature for 3 months or longer. In some embodiments, the mRNA maintains integrity of 80% or greater after storage at room temperature for 1 year or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at room temperature for 6 months or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at room temperature for 3 months or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at room temperature for 1 year or longer.
  • the mRNA maintains integrity of 80%, 85%, 90%, 92%, 94%, 96%, 98% or greater, including any values and subranges therebetween, after storage at 2- 8°C (e.g., 4°C) for 4 weeks or longer, such as 1 months, 3 months, 6 months, 9 months, or 1 year or longer, including any values and subranges therebetween.
  • the mRNA maintains integrity of 80% or greater after storage at 2-8°C (e.g., 4°C) for 4 weeks or longer.
  • the mRNA maintains integrity of 80% or greater after storage at 2-8°C (e.g., 4°C) for 3 months or longer.
  • the mRNA maintains integrity of 80% or greater after storage at 2-8°C (e.g., 4°C) for 6 months or longer. In some embodiments, the mRNA maintains integrity of 80% or greater after storage at 2- 8°C (e.g., 4°C) for a year or longer. In some embodiments, the mRNA maintains integrity of 85% or greater after storage at 2-8°C (e.g., 4°C) for 4 weeks or longer. In some embodiments, the mRNA maintains integrity of 85% or greater after storage at 2-8°C (e.g., 4°C) for 3 months or longer.
  • the mRNA maintains integrity of 85% or greater after storage at 2-8°C (e.g., 4°C) for 6 months or longer. In some embodiments, the mRNA maintains integrity of 85% or greater after storage at 2-8°C (e.g., 4°C) for a year or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at 2-8°C (e.g., 4°C) for 4 weeks or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at 2-8°C (e.g., 4°C) for 3 months or longer.
  • the mRNA maintains integrity of 90% or greater after storage at 2- 8°C (e.g., 4°C) for 6 months or longer. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at 2-8°C (e.g., 4°C) for a year or longer. In some embodiments, the mRNA maintains integrity of 95% or greater after storage at 2-8°C (e.g., 4°C) for 4 weeks or longer. In some embodiments, the mRNA maintains integrity of 95% or greater after storage at 2-8°C (e.g., 4°C) for 3 months or longer.
  • the mRNA maintains integrity of 95% or greater after storage at 2-8°C (e.g., 4°C) for 6 months or longer. In some embodiments, the mRNA maintains integrity of 95% or greater after storage at 2-8°C (e.g., 4°C) for a year or longer. [174] In some embodiments, the mRNA maintains integrity of 80% or greater after storage at -80 °C after 3 freeze thaw cycles. In some embodiments, the mRNA maintains integrity of 90% or greater after storage at -80 °C after 3 freeze thaw cycles.
  • the phrase “the mRNA maintains integrity of x% or greater after storage” means that the mRNA integrity does not decrease more than (100-x)% after storage.
  • mRNAs according to the present disclosure may be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Application No. US 2018/0258423, and can be used to practice the present disclosure, all of which are incorporated herein by reference. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT).
  • IVTT in vitro transcription
  • IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • a promoter e.g., a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • a buffer system that may include DTT and magnesium ions
  • an appropriate RNA polymerase e.g., T3, T7, or SP6 RNA polymerase
  • DNAse I e.g.,
  • a suitable mRNA sequence is an mRNA sequence encoding a protein or a peptide.
  • a suitable mRNA sequence is codon optimized for efficient expression human cells.
  • a suitable mRNA sequence is naturally-occurring or a wild-type sequence.
  • a suitable mRNA sequence encodes a protein or a peptide that contains one or mutations in amino acid sequence.
  • An exemplary mRNA coding sequence and the corresponding amino acid sequence are shown below:
  • X GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGA CACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGA UUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 1)
  • Y (3' UTR Sequence) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUU GCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGC U (SEQ ID NO: 2)
  • the mRNA comprises one or more nonstandard nucleotide residues.
  • the nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine (“5mC”), pseudouridine (“yU”), and/or 2-thio-uridine (“2sU”). See, e.g., U.S. Patent No. 8,278,036 or WO 2011/012316 for a discussion of such residues and their incorporation into mRNA.
  • the mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine.
  • RNA is disclosed US Patent Publication US 2012/0195936 and international publication WO 2011/012316, both of which are hereby incorporated by reference in their entirety.
  • the presence of nonstandard nucleotide residues may render an mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues.
  • the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5 -bromouracil, 5- propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6- aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications.
  • Some embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-O-alkyl modification, a locked nucleic acid (LNA)).
  • any of these modifications may be present in 0-100% of the nucleotides — for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100%, including any values and subranges therebetween, of the constituent nucleotides individually or in combination.
  • mRNAs may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(l-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
  • mRNAs may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'- fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'- deoxyuridine 5 '-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'- deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O- alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5'- triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligosineucle
  • a 5' cap and/or a 3' tail may be added after the synthesis.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • a 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’5’5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a 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. Additional cap structures are described in published U.S. Application No. US 2016/0032356 and published U.S. Application No. US 2018/0125989, which are incorporated herein by reference.
  • a poly A or poly C tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to
  • a tail structure includes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides, including any values and subranges therebetween.
  • the addition of the 5’ cap and/or the 3’ tail facilitates the detection of abortive transcripts generated during in vitro synthesis because without capping and/or tailing, the size of those prematurely aborted mRNA transcripts can be too small to be detected.
  • the 5’ cap and/or the 3’ tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA).
  • the 5’ cap and/or the 3’ tail are added to the synthesized mRNA before the mRNA is purified as described herein.
  • the 5’ cap and/or the 3’ tail are added to the synthesized mRNA after the mRNA is purified as described herein.
  • mRNA synthesized according to the present disclosure may be used without further purification.
  • mRNA synthesized according to the present disclosure may be used without a step of removing shortmers.
  • mRNA synthesized according to the present disclosure may be further purified.
  • Various methods may be used to purify mRNA synthesized according to the present disclosure. For example, purification of mRNA can be performed using centrifugation, filtration and /or chromatographic methods.
  • the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means.
  • the mRNA is purified by HPLC.
  • the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.
  • the mRNA is purified either before or after or both before and after capping and tailing, by centrifugation. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by filtration. In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing, by Tangential Flow Filtration (TFF). In some embodiments, the mRNA is purified either before or after or both before and after capping and tailing by chromatography.
  • TMF Tangential Flow Filtration
  • lipid nanoparticles comprise one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, lipid nanoparticles further comprise one or more cholesterol-based lipids. In some embodiments, the one or more cationic lipids constitutes about 30-70% of the total lipids in LNPs by molar %. In some embodiments, the one or more PEG-modified lipids constitutes about 1-15% of the total lipids in LNP by molar %. In some embodiments, the one or more non-cationic lipids constitutes about 10-40% of the total lipids in LNP by molar %. In some embodiments, the one or more cholesterol -based lipids comprise about 5-40% of the total lipids in LNP by molar %.
  • the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the lipid nanoparticles is approximately 60:25: 10:5. In some embodiments, the molar ratio of cationic lipid(s) to noncationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) in the lipid nanoparticles is approximately 40:25:30:5.
  • cationic lipids refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH.
  • Suitable cationic lipids for use in the compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2010/144740, which is incorporated herein by reference.
  • the compositions and methods of the present disclosure include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate, having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include ionizable cationic lipids as described in International Patent Publication WO 2013/149140, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid of one of the following formulas:
  • Ri and R2 are each independently selected from the group consisting of hydrogen, an optionally substituted, variably saturated or unsaturated C1-C20 alkyl and an optionally substituted, variably saturated or unsaturated C6-C20 acyl; wherein Li and L2 are each independently selected from the group consisting of hydrogen, an optionally substituted C1-C30 alkyl, an optionally substituted variably unsaturated C1-C30 alkenyl, and an optionally substituted C1-C30 alkynyl; wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., where m is three); and wherein n is zero or any positive integer (e.g., where n is one).
  • compositions and methods of the present disclosure include the cationic lipid (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12- dien-l-yl) tetracosa-15,18-dien-l-amine (“HGT5000”), having a compound structure of:
  • compositions and methods of the present disclosure include the cationic lipid (15Z, 18Z)- N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl) tetracosa-4, 15, 18-trien-l -amine (“HGT5001”), having a compound structure of:
  • compositions and methods of the present disclosure include the cationic lipid and (15Z, 18Z)- N,N-dimethyl-6-((9Z,12Z)-octadeca-9, 12-dien-l-yl) tetracosa-5,15,18-trien- 1 -amine (“HGT5002”), having a compound structure of:
  • compositions and methods of the disclosure include cationic lipids described as aminoalcohol lipidoids in International Patent Publication WO 2010/053572, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: (Cl 2-200) and pharmaceutically acceptable salts thereof.
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2016/118725, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2016/118724, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • Suitable cationic lipids for use in the compositions and methods of the disclosure include a cationic lipid having the formula of 14,25 -ditridecyl 15,18,21,24- tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publications WO 2013/063468 and WO 2016/205691, each of which are incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2015/184256, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • the X independently is O or S
  • each Y independently is O
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2016/004202, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure: or a pharmaceutically acceptable salt thereof.
  • compositions and methods of the present disclosure include cationic lipids as described in United States Provisional Patent Application Serial Number 62/758,179, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • each R 1 and R 2 is independently H or Ci-Ce aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or arylene; each L 1 is independently an ester, thioester, disulfide, or anhydride group; each L 2 is independently C2-C10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C6-C20 aliphatic.
  • the compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • Suitable cationic lipids for use in the compositions and methods of the present disclosure include the cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which is incorporated herein by reference.
  • the cationic lipids of the compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2015/199952, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include ; cationic lipid having the compound structure: [227] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include ; cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include ; cationic lipid having the compound structure: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2017/004143, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the [238] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the [242] and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure:
  • compositions and methods of the present disclosure include a cationic lipid having the
  • compositions and methods of the present disclosure include a cationic lipid having the and pharmaceutically acceptable salts thereof.
  • Suitable cationic lipids for use in the compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2017/117528, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having the compound structure: and pharmaceutically acceptable salts thereof.
  • the compositions and methods of the present disclosure include a cationic lipid having the compound structure: and pharmaceutically acceptable salts thereof.
  • Suitable cationic lipids for use in the compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2017/049245, which is incorporated herein by reference.
  • the cationic lipids of the compositions and methods of the present disclosure include a compound of one of the following formulas: and pharmaceutically acceptable salts thereof.
  • R4 is independently selected from -(CH2) n Q and
  • Q is selected from the group consisting of -OR, -OH, -O(CH2) n N(R)2, - OC(O)R, -CX 3 , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) 2 R, -N(H)S(O) 2 R, - 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 1, 2, or 3.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • Suitable cationic lipids for use in the compositions and methods of the disclosure include the cationic lipids as described in International Patent Publication WO 2017/173054 and WO 2015/095340, each of which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of: and pharmaceutically acceptable salts thereof.
  • compositions and methods of the present disclosure include a cationic lipid having a compound structure of:
  • compositions and methods of the present disclosure include cleavable cationic lipids as described in International Patent Publication WO 2012/170889, which is incorporated herein by reference.
  • the compositions and methods of the present disclosure include a cationic lipid of the following formula: wherein Ri is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl; wherein R2 is selected from the group consisting of one of the following two formulas: and wherein R3 and R4 are each independently selected from the group consisting of an optionally substituted, variably saturated or unsaturated C6-C20 alkyl and an optionally substituted, variably saturated or unsaturated C6-C20 acyl; and wherein n is zero or any positive integer (e.g
  • compositions and methods of the present disclosure include a cationic lipid, “HGT4001”, having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid, “HGT4002”, having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid, “HGT4003”, having a compound structure of:
  • compositions and methods of the present disclosure include a cationic lipid, “HGT4004”, having a compound structure of:
  • compositions and methods of the present disclosure include HEPES-based disulfide cationic lipids with a piperazine core as described in International Patent Publication WO 2022/221688, which is incorporated herein by reference.
  • compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include a cationic lipid of the following formula: (GL-HEPES-E3 -E 12-DS-4-E10 : (2-(4-(2-((3 -(bi s(2- hydroxydecyl)amino)butyl)disulfaneyl) ethyl)piperazin- 1 -yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate)) and pharmaceutically acceptable salts thereof.
  • the compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include cationic lipids as described in Dong et al., PNAS, 2014, 111(11):3955- 3960 and U.S. Pat. No. 9,512,073, which are incorporated herein by reference.
  • the compositions and methods of the present disclosure include a cationic lipid of the following formula:
  • compositions and methods of the present disclosure include cleavable cationic lipids as described in U.S. Provisional Application No. 62/672,194, filed May 16, 2018, and incorporated herein by reference.
  • the compositions and methods of the present disclosure include a cationic lipid that is any of general formulas or any of structures (la)-(21a) and (lb)-(2 lb) and (22)- (237) described in U.S. Provisional Application No. 62/672,194.
  • compositions and methods of the present disclosure include a cationic lipid that has a structure according to Formula (I’), wherein:
  • R x is independently -H, -l R 1 , or -L 5A -L 5B -B’; each of L 1 , L 2 , and L 3 is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or - C(0)NR L -; each L 4A and L 5A is independently -C(O)-, -C(O)O-, or -C(0)NR L -; each L 4B and L 5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene; each B and B’ is NR 4 R 5 or a 5- to 10-membered nitrogen-containing heteroaryl; each R 1 , R 2 , and R 3 is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; each R 4 and R 5 is independently hydrogen, C1-C10 alky
  • compositions and methods of the present disclosure include a cationic lipid that is Compound (139) of 62/672,194, having a compound structure of:
  • compositions and methods of the present disclosure include the cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • cationic lipids suitable for the compositions and methods of the present disclosure include, for example, 5- carboxyspermylglycinedioctadecylamide (“DOGS”); 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (“DOSPA”) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-Dioleoyl- 3 -Dimethylammonium -Propane (“DODAP”); l,2-Dioleoyl-3 -Trimethylammonium -
  • DOGS 5- carboxyspermylglycinedioctadecylamide
  • DOSPA 2,3-dioleyloxy-N-[2(spermine- carboxa
  • Additional exemplary cationic lipids suitable for the compositions and methods of the present disclosure also include: l,2-distearyloxy-N,N-dimethyl-3-aminopropane ( “DSDMA”); l,2-dioleyloxy-N,N-dimethyl-3 -aminopropane (“DODMA”); 1 ,2- dilinoleyloxy-N,N-dimethyl-3 -aminopropane (“DLinDMA”); l,2-dilinolenyloxy-N,N- dimethyl-3 -aminopropane (“DLenDMA”); N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
  • one or more cationic lipids suitable for the compositions and methods of the present disclosure include 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,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- tri s(3 -oxo-3 -(undecylamino)propyl)-N 1 ,N16-diundecyl-4,7, 10,13 -tetraazah exadecane- 1,16-diamide (“NC98-5”).
  • XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-
  • compositions and methods of the present disclosure include the cationic lipid known as ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,l- diyl)bis(2-hexyldecanoate)), which is a synthetic lipid having the following chemical structure: and pharmaceutically acceptable salts thereof.
  • ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,l- diyl)bis(2-hexyldecanoate)
  • compositions of the present disclosure include 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 subranges therebetween, measured by weight, of the total lipid content in the composition, e.g., a lipid nanoparticle.
  • the compositions of the present disclosure include one or more cationic lipids that constitute about 30-70 % (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 values and subranges therebetween), measured as mol %, of the total lipid content in the composition, e.g., a lipid nanoparticle.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (
  • a non-cationic lipid is DPPC. In some embodiments, a noncationic lipid is DOPE. In some embodiments, a non-cationic lipid is DEPE.
  • non-cationic lipids may be used alone, but are preferably used in combination with other lipids, for example, cationic lipids.
  • the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10 % to about 70% of the total lipid present in a liposome.
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • provided liposomes comprise one or more cholesterol-based lipids.
  • suitable cholesterol-based lipids include cholesterol and, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.
  • DC-Choi N,N-dimethyl-N-ethylcarboxamidocholesterol
  • l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat.
  • PEG polyethylene glycol
  • PEG-CER derivatized ceramides
  • C8 PEG-2000 ceramide N-Octanoyl-Sphingosine-1- [Succinyl(Methoxy Polyethylene Glycol)-2000]
  • C8 PEG-2000 ceramide is also contemplated by the present disclosure, either alone or preferably in combination with other lipid formulations together which comprise the transfer vehicle (e.g., a lipid nanoparticle).
  • a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein.
  • liposomal delivery vehicles as used herein, also encompass nanoparticles comprising polymers.
  • Suitable polymers may include, for example, polyacrylates, poly alky cyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI).
  • PEI polyethylenimine
  • cationic lipids non-cationic lipids and/or PEG-modified lipids which comprise the lipid mixture as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s) and the nature of the and the characteristics of the mRNA to be encapsulated. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
  • the LNPs are manufactured with a particular molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids.
  • lipid nanoparticle comprises a molar ratio of cationic lipids to noncationic lipids to cholesterol-based lipids to PEG-modified lipids of 60:25:10:5.
  • lipid nanoparticle comprises a molar ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids of 40:25:30:5.
  • a formulation described herein comprises mRNA encoding for a drug or a peptide or protein therapeutic suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for any peptide or polypeptide suitable for the present disclosure.
  • a formulation is a dry powder formulation (e.g., a reconstitutable dry powder formulation).
  • a formulation is a reconstituted formulation (e.g., reconstituted from a reconstitutable dry powder formulation as described herein).
  • a formulation described herein comprises other nucleic acids for in vivo administration, as discussed herein.
  • a formulation described herein comprises a full-length mRNA that encodes for any antibody suitable for the present disclosure. In some embodiments, a formulation described herein comprises a full-length mRNA that encodes for any therapeutic protein suitable for the present disclosure. In some embodiments, a formulation described herein comprises a full-length mRNA that encodes for any naturally occurring peptide suitable for the present disclosure. In some embodiments, a formulation described herein comprises a full-length mRNA that encodes for any modified or non-naturally occurring peptide suitable for the present disclosure. In some embodiments, a formulation described herein comprises a full-length mRNA that encodes for any peptide drug suitable for the present disclosure.
  • an mRNA encodes one or more naturally occurring peptides. In some embodiments, an mRNA encodes one or more modified or non-natural peptides.
  • the peptide drug include, glucose-dependent insulinotropic polypeptide.
  • a further example of the peptide drug is elamipretide.
  • Further examples of the peptide drug are cyclotides (which are peptides characterized by their head-to-tail cyclized peptide backbone and the interlocking arrangement of their disulfide bonds), including, e.g., a cyclotide having at least two disulfide bonds (and preferably a cyclotide having three disulfide bonds).
  • the peptide drug is selected from GLP-1, amylin, an amylin analog, pramlintide, a somatostatin analog (e.g., octreotide, lanreotide, or pasireotide), goserelin (e.g., goserelin acetate), buserelin, peptide YY (PYY), a PYY analog, glatiramer (e.g., glatiramer acetate), leuprolide (e.g., leuprolide acetate), desmopressin (e.g., desmopressin acetate, particularly desmopressin monoacetate trihydrate), teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, bortezomib, cosyntropin, sermorelin, luteinizing-hormone-releasing hormone (LHRH),
  • GLP-1 amylin
  • a formulation described herein comprises a full-length mRNA that encodes for any peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for any peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with a urea cycle disorder suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with a lysosomal storage disorder suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with a glycogen storage disorder suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with amino acid metabolism suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with a lipid metabolism or fibrotic disorder suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a protein associated with methylmalonic acidemia suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a peptide or polypeptide for use in the delivery to or treatment of the cardiovasculature of a subject or a cardiovascular cell suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for any peptide or polypeptide for use in the delivery to or treatment of the muscle of a subject or a muscle cell suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for a peptide or polypeptide for use in the delivery to or treatment of the nervous system of a subject or a nervous system cell suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for any peptide or polypeptide for use in the delivery to or treatment of the eye of a subject or a eye cell suitable for the present disclosure.
  • Exemplary embodiments are described herein. a. Liver
  • the present disclosure provides a formulation having full- length mRNA for delivery to the liver.
  • the dry powder formulation is reconstituted.
  • the present disclosure provides a formulation having full- length mRNA for delivery to liver or a formulation having full-length mRNA for treatment of liver-associated condition.
  • the present disclosure provides a formulation having full-length mRNA that encodes for ATP7B protein, also known as Wilson disease protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for porphobilinogen deaminase enzyme.
  • the present disclosure provides a formulation having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.
  • the present disclosure provides a formulation having full-length mRNA that encodes for human hemochromatosis (HFE) protein.
  • HFE human hemochromatosis
  • a formulation described herein comprises a full-length mRNA that encodes for a peptide or polypeptide for use in the delivery of or treatment into the lung of a subject or a cell of a subject suitable for the present disclosure. Additional embodiments are described herein.
  • the present disclosure provides a formulation comprising full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the lung of a subject or a lung cell.
  • the present disclosure provides a formulation having full- length mRNA that encodes for cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for ATP -binding cassette sub-family A member 3 protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for dynein axonemal intermediate chain 1 protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for dynein axonemal heavy chain 5 (DNAH5) protein.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the present disclosure provides a formulation having full-length mRNA that encodes for alpha- 1 -antitrypsin protein. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for forkhead box P3 (F0XP3) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes one or more surfactant protein, e.g., one or more of surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein. In some embodiments, dry powder formulations comprising trehalose are used for lung delivery. In some embodiments, the formulations comprising a sugar or sugar alcohol excipients (for example, trehalose) having a particle size less than 5 microns are suited to lung delivery. c. Heart
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiovasculature of a subject or a cardiovascular cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for vascular endothelial growth factor A protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for relaxin protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for bone morphogenetic protein-9 protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for bone morphogenetic protein-2 receptor protein.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the muscle of a subject or a muscle cell. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for dystrophin protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for frataxin protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the cardiac muscle of a subject or a cardiac muscle cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for a protein that modulates one or both of a potassium channel and a sodium channel in muscle tissue or in a muscle cell. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for a protein that modulates a Kv7.1 channel in muscle tissue or in a muscle cell. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for a protein that modulates a Navi.5 channel in muscle tissue or in a muscle cell. e. Nerve cells
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the nervous system of a subject or a nervous system cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for survival motor neuron 1 protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for survival motor neuron 2 protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for frataxin protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for ATP binding cassette subfamily D member 1 (ABCD1) protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for CLN3 protein.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the blood or bone marrow of a subject or a blood or bone marrow cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for beta globin protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for Bruton's tyrosine kinase protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for one or clotting enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the kidney of a subject or a kidney cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for collagen type IV alpha 5 chain (COL4A5) protein.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the eye of a subject or an eye cell.
  • the present disclosure provides a formulation having full-length mRNA that encodes for ATP -binding cassette sub-family A member 4 (ABCA4) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for retinoschisin protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for retinal pigment epithelium-specific 65 kDa (RPE65) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for centrosomal protein of 290 kDa (CEP290).
  • ABCA4 ATP -binding cassette sub-family A member 4
  • a formulation described herein comprises a full-length mRNA that encodes for a peptide or polypeptide for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject suitable for the present disclosure. Additional embodiments are described herein.
  • a formulation described herein comprises a full-length mRNA that encodes for an antigen (e.g., from an infectious agent such as a virus) suitable for the present disclosure.
  • a formulation described herein comprises a full- length mRNA that encodes for an immunomodulator suitable for the present disclosure.
  • a formulation described herein comprises a full-length mRNA that encodes for an endonuclease suitable for the present disclosure.
  • Cancer is considered an immunological disease, and cancer immunotherapy has become the center-stage of research and development of the present day.
  • vaccines are developed to boost the immune system to turn against cancer antigens and eliminate a tumor by activated cytotoxic T cells directed against the antigens.
  • Epstein Barr Virus (EBV) antigens such as EBV1 and EBV2 associated with lymphoma and nasopharyngeal carcinoma
  • EBV Epstein Barr Virus
  • HPV Human Papilloma Virus
  • HPV Human Papilloma Virus
  • HPV Human Papilloma Virus
  • HCC Hepatitis B Virus
  • HCV Hepatitis C Virus
  • HCC human T lymphotropic virus type 1
  • HHV-8 human herpes virus 8 with Kaposi sarcoma
  • cancerous cells express antigens that are not commonly expressed by non-cancerous cells or tissues.
  • antigens include but are not limited to epithelial tumor antigen (ETA) found in breast cancer, RAS family member p-53 and other activated RAS antigens, ovarian cancer antigens BRCA1 and BRCA2, melanoma associated antigen (MAGE) found in malignant melanoma cells, BCR-ABL fusion gene product found in myeloid leukemia, acute lymphoblastic leukemia, acute myelogenic leukemia, BRAF antigens found in cutaneous melanoma and colorectal cancer, epithelial growth factor receptor (EGFR) for 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, NY-ESO 1 found in neuroblastoma, Melanoma-associated antigen recognized by T cells (MART).
  • ETA epit
  • cancer antigens being endogenous are not presented by antigen presenting cells (APCs) in a manner similar to viral antigens, i.e., in association with the MHC-1 molecule categorizing the antigen as foreign, but cytotoxic T cells are able to differentiate and identify mutated selfantigens and possess an inherent property to seek and destroy cells that bear the mutated antigens. Therefore, the current objective of cancer immunotherapy is to achieve optimum activation of cytotoxic T cells directed against the mutated antigens.
  • a patient’s specific mutations associated with her/his cancer can be mapped and used to generate vaccines, inducing the patient’s own cytotoxic T cells to generate the necessary immune response to destroy tumor cells.
  • mRNA vaccines could be the safe and cost-effective alternative to peptide vaccines for enabling such personalized medicine.
  • Pan-genomic scanning and analysis of mutations present in a cancer patient could be used to specifically design mRNA encoding an antigen or epitope containing the mutation, which when administered in vivo will produce the translated product on a cell surface. This would direct an immune response against the mutated antigen. In the process, cytotoxic T cells attack the tumor cells, which inherently express the mutated antigen.
  • Methods and protocols involved in executing pan genomic sequencing analysis, mutation analysis, epitope mapping and analysis and designing suitable peptides for vaccination are known to one of skill in the art.
  • mRNA vaccines bypass the HLA-matching for the receiving host.
  • a pathogen proteome can be scanned for antigenic signatures with vaccine potential.
  • Proteome database can be accessed using Uniprot Consortium, uniprot.org/). This could be effective in new pathogens, such as Zika virus. This type of reverse vaccinology has been employed in identifying a number of novel peptide vaccine candidates. New peptide vaccines were also identified from Helicobacter pylori and Mycobacterium tuberculosis by combining genomics and proteomics (See, for example, Etz et al., PNAS. 2002, 99 (10) 6573-6578).
  • Whether the potential antigenic candidate can generate successful immune response can be verified by suitably expressing a library of potential antigens by various forms of cell surface display and subjecting to testing opsonization and antibody binding.
  • Exemplary 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, immunet.cn/ced/index.php; HIV database:hiv. lanl.gov/content/immunology for immunogenetics and immunoinformatics.
  • mRNA vaccines offer many advantages over present cell-based vaccines using live, attenuated or killed pathogen or toxoid vaccines.
  • the dry powder formulation of the present disclosure can used to deliver mRNA for vaccination.
  • mRNA vaccines are cost-effective and provide flexible design platform.
  • mRNA encoding an antigen could be directed to induce specific immune response, and therefore can be applied in developing a wide range of therapeutic and prophylactic mRNA vaccines for a wide variety of diseases, including infections and cancers.
  • Vaccine candidates are well established for a large number of infectious pathogens.
  • vaccines are agents that mimic at least in part a disease-causing agent and thereby elicit an immune response by the mammalian host.
  • vaccines are biological agents, such as heat killed, irradiated or otherwise attenuated pathogenic organisms, live attenuated microbes, protein or peptide antigens, conjugated antigens, toxins or microbial surface proteins or fragments thereof.
  • an mRNA encoding a protein or a peptide antigen is a safe and effective way to induce an immune response against the disease.
  • mRNA can be effectively delivered to express in vivo by encapsulated in a liposome comprising suitable lipids discussed in a later section.
  • This mRNA encoding the antigenic peptide or protein could therefore be used to generate the vaccine in vivo.
  • An immune response generated by the mammalian host against the vaccine component is intended in turn to protect the host from a subsequent attack by the pathogen, since the immune system of the host is primed for the attack by the pathogen.
  • the host system has immunological memory (a component of the adaptive immune response) of the pathogen. This process is known as prophylactic vaccination.
  • a vaccine may boost the host’s immune system in an existing infection, for example by redirect an immune response against new and less recognized microbial antigen(s) (subdominant antigens) which then induce a strong immune response leading to pathogen elimination.
  • This type of vaccine response may be categorized as therapeutic vaccination.
  • Pattern recognition molecules include a variety of germline-encoded receptors specialized in discriminating between microbial and host cell surfaces, or infected and normal cells. Phagocytes (monocytes, macrophages and dendritic cells), express pattern recognition molecules on their surface and are primarily responsible for recognizing, killing and elimination of the pathogens in an innate immune response.
  • phagocytes also process and present antigens to the circulating lymphocytes for generating a more specific antigen-targeted immune response, also known as the adaptive immune response.
  • activated lymphocytes mature in the lymph nodes into antigen-specific T-cells expressing receptors for recognition of the antigen such that effector cytotoxic T cells recognize and kill a cell expressing the antigen when present in association with a second set of cell-surface molecules, the Major Histocompatibility Complex molecules or MHC; and helper T cells activate the system to generate the T cell memory and the humoral immune response.
  • the humoral immune response comprises antibody-secreting B cells generated by clonal expression and differentiation over the course of several days, during which time that innate immunity continues to function.
  • cytotoxic T cells Clonal expansion of cytotoxic T cells also occurs rapidly in lymphoid organs, such as lymph nodes and is augmented by exposure to antigens.
  • Activated T cells generate a number of cytokines, such as Interferon Gamma (IFN-y) and Tumor Necorsis Factor alpha (TNF-a) which are considered the hallmarks of T cell activation.
  • IFN-y Interferon Gamma
  • TNF-a Tumor Necorsis Factor alpha
  • a successful vaccine generates a rapid and robust cytotoxic T cell response, a strong antibody response and a lasting immunological memory.
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with a lipid metabolism or fibrotic disorder. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for an mTOR inhibitor. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for ATPase phospholipid transporting 8B1 (ATP8B1) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for one or more NF-kappa B inhibitors, such as one or more of I-kappa B alpha, interferon-related development regulator 1 (IFRD1), and Sirtuin 1 (SIRT1).
  • IFRD1 interferon-related development regulator 1
  • SIRT1 Sirtuin 1
  • the present disclosure provides a formulation having full-length mRNA that encodes for PPAR-gamma protein or an active variant.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from an infectious agent, such as a virus.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from influenza virus.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from respiratory syncytial virus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from rabies virus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from cytomegalovirus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from rotavirus.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from human papillomavirus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from a human metapneumovirus.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from malaria virus.
  • the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from zika virus. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen from chikungunya virus. [335] In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antigen determined from a subject's own cancer cell, i.e., to provide a personalized cancer vaccine.
  • the present disclosure provides a formulation having full- length mRNA that encodes for an antibody.
  • the antibody can be a bispecific antibody. In some embodiments, the antibody can be part of a fusion protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antibody to 0X40. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antibody to VEGF. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antibody to tissue necrosis factor alpha. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antibody to CD3. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an antibody to CD 19.
  • the present disclosure provides a composition having full- length mRNA that encodes for an immunomodulator. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for Interleukin 12. In some embodiments, the present disclosure provides a composition having full-length mRNA that encodes for Interleukin 23. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for Interleukin 36 gamma. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for a constitutively active variant of one or more stimulator of interferon genes (STING) proteins.
  • STING interferon genes
  • the present disclosure provides a composition having full- length mRNA that encodes for an endonuclease. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for an RNA-guided DNA endonuclease protein, such as Cas 9 protein. In some embodiments the present disclosure provides a formulation having full-length mRNA that encodes for a meganuclease protein. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for a transcription activator-like effector nuclease protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for a zinc finger nuclease protein.
  • the present disclosure provides a formulation comprising full- length mRNA that encodes a secreted protein. In some embodiments, the present disclosure provides a formulation comprising full-length mRNA that encodes a nuclear protein. In some embodiments, the present disclosure provides a formulation comprising full-length mRNA that encodes a metabolic protein. In some embodiments, the present disclosure provides a formulation comprising full-length mRNA that encodes a cytoplasmic protein. In some embodiments, the present disclosure provides a composition comprising full-length mRNA that encodes a membrane protein. In some embodiments, the present disclosure provides a formulation comprising full-length mRNA that encodes a mitochondrial protein.
  • the present disclosure provides a formulation comprising full-length mRNA that encodes a lysosomal protein.
  • an mRNA encodes a cytosolic protein.
  • an mRNA encodes a protein associated with the actin cytoskeleton.
  • an mRNA encodes a protein associated with the plasma membrane.
  • the present disclosure provides a formulation having full- length mRNA that encodes a peptide or polypeptide for use in the delivery to or treatment of the liver of a subject or a liver cell.
  • peptides and polypeptides can include those associated with a urea cycle disorder, associated with a lysosomal storage disorder, with a glycogen storage disorder, associated with an amino acid metabolism disorder, associated with a lipid metabolism or fibrotic disorder, associated with methylmalonic acidemia, or associated with any other metabolic disorder for which delivery to or treatment of the liver or a liver cell with enriched full-length mRNA provides benefit.
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with a urea cycle disorder. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for ornithine transcarbamylase (OTC) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for arginosuccinate synthetase 1 protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for carbamoyl phosphate synthetase 1 protein.
  • OTC ornithine transcarbamylase
  • the present disclosure provides a formulation having full-length mRNA that encodes for arginosuccinate lyase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for arginase protein.
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with methylmalonic acidemia.
  • the present disclosure provides a formulation having full- length mRNA that encodes for methylmalonyl CoA mutase protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for methylmalonyl CoA epimerase protein.
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with a lysosomal storage disorder. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for alpha galactosidase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for glucocerebrosidase protein. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for iduronate-2-sulfatase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for iduronidase protein.
  • the present disclosure provides a therapeutic formulation having full-length mRNA that encodes for N-acetyl-alpha-D-glucosaminidase protein. In some embodiments, the present disclosure provides a formulation having full- length mRNA that encodes for heparan N-sulfatase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for galactosamine-6 sulfatase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for beta-galactosidase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for lysosomal lipase protein.
  • the present disclosure provides a formulation having full-length mRNA that encodes for arylsulfatase B (N- acetylgalactosamine-4-sulfatase) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for transcription factor EB (TFEB).
  • TFEB transcription factor EB
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with a glycogen storage disorder. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for acid alpha-glucosidase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for glucose-6-phosphatase (G6PC) protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for liver glycogen phosphorylase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for muscle phosphoglycerate mutase protein. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for glycogen debranching enzyme.
  • G6PC glucose-6-phosphatase
  • the present disclosure provides a formulation having full-length mRNA that encodes for liver glycogen phosphorylase protein.
  • the present disclosure provides a formulation having full
  • the present disclosure provides a formulation having full- length mRNA that encodes for a protein associated with amino acid metabolism. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for phenylalanine hydroxylase enzyme. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for glutaryl-CoA dehydrogenase enzyme. In some embodiments, the present disclosure provides a formulation having full-length mRNA that encodes for propionyl-CoA carboxylase enzyme. In some embodiments the present disclosure provides a formulation having full-length mRNA that encodes for oxalase alanine-glyoxylate aminotransferase enzyme.
  • the present disclosure provides a formulation comprising a nucleic acid, including, for example, a therapeutic nucleic acid, including, but not limited to a plasmid, a viral vector, an antisense nucleic acid, an siRNA, microRNA, ribozymes, antagomirs, aptamers, CRISPR nucleic acids (e.g., guide RNA, crRNA, or tracr RNA), nucleic acids for gene therapy, nucleic acids for DNA editing, probes, or any other oligonucleotide that is susceptible to degradation by nucleases and/or harsh environmental conditions (e.g., pH), including other oligonucleotides that are to be administered in vivo.
  • a nucleic acid including, for example, a therapeutic nucleic acid, including, but not limited to a plasmid, a viral vector, an antisense nucleic acid, an siRNA, microRNA, ribozymes, antagomirs, aptamers, C
  • compositions described herein can be administered according to various methods known in the art, whether as a dry powder formulation or following reconstitution.
  • the pharmaceutical formulations of the disclosure may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue, including sustained release formulations.
  • dry powder formulations and/or reconstituted dry powder formulations of the present invention may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the nature of the encapsulated materials, the site and method of administration, the scheduling of administration, the subject’s age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.
  • Suitable routes of dry powder formulations and/or reconstituted dry powder formulations disclosed herein include, for example, oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, intracerebroventricular. direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections or infusions.
  • Dry powder formulations and/or reconstituted dry powder formulations of the present invention may be administered in a local rather than systemic manner.
  • a reconstituted dry powder formulation may be administered via injection or infusion of the pharmaceutical compositions directly into a targeted tissue, preferably in a depot or sustained release formulation, such that the contacting of the targeted cells with the constituent lipid nanoparticles may further facilitated.
  • Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • Formulations of the present invention complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, such compositions can be applied surgically without the use of polymers or supports.
  • Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • tissue in which delivered mRNA may be delivered and/or expressed include, but are not limited to the lungs, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.
  • the tissue to be targeted in the liver For example, aerosols containing compositions of the present disclosure can be inhaled (for nasal, tracheal, or bronchial delivery). ii. Delivery to the Airways
  • compositions of the present disclosure can be delivered using a metered dose inhaler. In some embodiments, compositions of the present disclosure can be reconstituted and nebulized for delivery. In some embodiments, compositions of the present disclosure can be injected into the site of injury, disease manifestation, or pain. In some embodiments, compositions of the present disclosure can be provided for oral, tracheal, or esophageal applications.
  • a formulation of the present disclosure is reconstituted into a liquid solution and nebulized for delivery.
  • Nebulization can be achieved by any nebulizer known in the art.
  • a nebulizer transforms a liquid to a mist so that it can be inhaled more easily into the lungs.
  • Nebulizers are effective for infants, children and adults. Nebulizers are able to nebulize large doses of inhaled medications.
  • a nebulizer for use with the disclosure comprises a mouthpiece that is detachable.
  • Therapeutic treatments for liver and lung diseases are being developed by delivering synthetic mRNA encoding a missing and/or a non-functional protein to the corresponding organ cells via intravenous and inhalable routes, respectively.
  • the particles of the formulation affect distribution and deposition of an aerosol within the respiratory system.
  • particle deposition to the large conducting airways is preferred for effective absorption and distribution of the therapeutic component.
  • Aerosol of very fine particles for instance, particles having less than 1 micrometer diameter may be deposited peripherally for effective absorption by specific cells of the lung, such as smooth muscles for an active pharmaceutical ingredient functioning as bronchodilator.
  • formulations comprising trehalose are used for lung delivery.
  • the formulations comprising a sugar or sugar alcohol excipients (for example, trehalose) having a particle size less than 5 microns are suited to lung delivery.
  • compositions of the present disclosure can be supplied in reconstituted liquid, powder, tablet, granules, implants, or capsule form for administration to the stomach or intestines.
  • the pharmaceutical composition is in the form of a tablet with an enteric coating.
  • the enteric coating comprises: hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, cellulose ester-ether phthalate, hydroxypropylcellulose phthalate, alkali salts of cellulose acetate phthalate, alkaline earth salts of cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, carboxymethylcellulose sodium, acrylic acid polymers and copolymers, ethyl acrylate/methyl methacrylate/ethyl trimethylammonium chloride methacrylate terpolymer, methacrylic acid/ethyl acrylate copolymer, methacrylic acid/methyl methacrylate copolymer, polyvinyl pyr
  • the tablets are manufactured using filler-binders for direct compaction and to promote cohesiveness.
  • Some exemplary binders include Polyvinyl Pyrrolidone (PVP), Methylcellulose, Hydroxy Propyl Methyl Cellulose (HPMC), Polymethacrylates, Sodium Carboxy Methyl Cellulose, Polyethylene Glycol (PEG) and Methylcellulose, Sucrose, Acacia, Methyl Cellulose, Liquid glucose, Tragacanth, Ethyl Cellulose, Gelatin, Starch Paste, Hydroxy Propyl Cellulose, Pregelatinized Starch, Sodium Carboxy Methyl Cellulose, Alginic Acid, Polyvinyl Alcohols, Polymethacrylates. iv. Other routes of administration
  • compositions of the present disclosure can be supplied for rectal or vaginal application. In some embodiments, compositions of the present disclosure can be delivered to the eye as drops, or even intravitreal or intraocular injection.
  • compositions of the present disclosure can be used for any parenteral and mucosal routes.
  • liquid formulation can be administered by for example, by infusion, perfusion, administration using a pen injector, cartridge system needle-array or patch and/or administration by a catheter system.
  • the administration is by subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection, or topical administration. There are many common forms of topical medication such as lotions, gels, patches, and powders, creams and ointments.
  • the parenteral administration includes buccal, sublingual, palatal, gingival, nasal, vaginal, cervical, rectal, or transdermal administration.
  • parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, and intraventricular.
  • methods of administration may further include oral administration.
  • administration orally may include a mouthwash, a mouth rinse, an oral rinse, a mouth bath, and the like.
  • cervical and/or vaginal mucosa administration may be via a solution, a gel, a suspension, a cream, an ointment, a foam, a pessary, or a tablet.
  • the reconstituted composition may be administered to the cervical and/or vaginal mucosa of a subject.
  • the cervical and/or vaginal mucosa administration may be via a solution, gel, suspension, cream, ointment, foam, pessary, or tablet.
  • the formulation or the reconstituted formulation is suitable for mucosal delivery.
  • the formulation is suitable for oral delivery.
  • the formulation is suitable for sublingual delivery.
  • the formulation is suitable for or intranasal delivery.
  • the formulation is suitable for buccal delivery.
  • the formulation is suitable for intramuscular.
  • the formulation is suitable for intravenous.
  • the formulation is suitable for subcutaneous.
  • a dry powder formulation for reconstitution comprising
  • lipid nanoparticle comprising messenger RNA (mRNA) encapsulated by one or more lipids; and wherein the w/w ratio of the excipient of (a) and the total lipids in the LNP of (b) is at least about 5.
  • any one of numbered embodiments 1-5 comprising an LNP that comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • a dry powder formulation for reconstitution comprising
  • lipid nanoparticle comprising messenger RNA (mRNA) encapsulated by one or more lipids; and. wherein the w/w ratio of the excipient of (a) and the total lipids in the LNP of (b) is at least about 5.
  • a method of delivering mRNA in vivo comprising administering to a subject in need of a reconstituted dry powder formulation of any one of numbered embodiments 1-27.
  • a method of preparing a dry powder formulation for reconstitution comprising:
  • a method of preparing a reconstituted dry powder formulation comprising (a) providing a dry powder formulation prepared according to numbered embodiment 31 ;
  • a dry powder formulation for reconstitution comprising
  • lipid nanoparticle comprising messenger RNA (mRNA) encapsulated by one or more lipids; and. wherein the w/w ratio of the excipient of (a) and the total lipids in the LNP of (b) is at least about 5.
  • sucrose sucrose. 3. The dry powder formulation of embodiment 1 or 2, wherein the w/w ratio of the sugar or the sugar alcohol to the total lipids is at least about 5.6, 11 , or 15, optionally wherein the w/w ratio of the sugar or the sugar alcohol to the total lipids is at least about 11.1 or at least about 15.6.
  • parenteral delivery optionally wherein the formulation is suitable for intramuscular, intravenous, or subcutaneous delivery; and/or
  • a method of delivering mRNA in vivo comprising administering to a subject in need of a reconstituted dry powder formulation of any one of embodiments 1-12, optionally wherein the reconstituted dry powder formulation is administered subcutaneously, intravenously, or intramuscularly.
  • a method of treating a disease or disorder in a subject by administering to the subject a reconstituted dry powder formulation of any one of embodiments 1-13, optionally wherein the reconstituted dry powder formulation is administered subcutaneously, intravenously, or intramuscularly.
  • a method of preparing a dry powder formulation for reconstitution comprising:
  • sucrose or trehalose wherein the concentration of sucrose or trehalose is greater than 2% (w/v), greater than 5% (w/v), or greater than 7% (w/v) post-reconstitution; and/or
  • Target mRNAs were synthesized by in vitro transcription employing RNA polymerase with a plasmid DNA template encoding the gene using unmodified nucleotides. This was followed by the addition of a 5’ cap structure (Cap 1) and a 3’ poly(A) tail.
  • An ethanolic solution of a mixture of lipids ionizable lipid, phosphatidylethanolamine, cholesterol and polyethylene glycol-lipid
  • mRNA-LNP suspensions were formulated into a final diluent containing different sugar-alcohol and sugar excipients at their desired concentration in 20% ethanol.
  • the resultant mRNA-LNP suspension was spray dried on a Buchi-290 spray dryer using parameters as shown in Table 1. Varying excipient amounts as shown in Table 2 and Table 3 were evaluated to identify the optimum excipient to total lipid amount ratio to generate a stable reconstituted product.
  • FIG. 2B shows the appearances of reconstituted dry powder with different amounts of mannitol as excipient (left: 7% mannitol (w/v); middle: 5% mannitol (w/v); right 2.5% mannitol (w/v)).
  • mannitol as excipient
  • iii. Sucrose [371] Although the DPP particle size was bigger, the product reconstituted well at and above sucrose to lipid ratio of 11.1. Although the DPP at sucrose/lipid ratio of 5.6 also reconstituted, the LNP particle size of the reconstituted product was larger (127 nm). The yield of the DPP was about 30% at and above sucrose/lipid weight ratio of 11.1.
  • FIG. 3A shows the dry powder characteristics and post reconstitution composition and characteristics with sucrose as the sugar excipient.
  • FIG. 3A shows no aggregates were observed using a microscope for reconstituted dry powder with 5% sucrose (w/v) as excipient as compared to water alone.
  • FIG. 3B shows the appearances of reconstituted dry powder with different amounts of sucrose as excipient (left: 7% sucrose (w/v); middle: 5% sucrose (w/v); right 2.5% sucrose (w/v)).
  • FIG. 3C shows mRNA-LNP formulation with 2.5% sucrose (w/v) as excipient did not spray dry well and the liquid formulation adhered to the cyclone separator.
  • the dry powder particle size was under 5 micron for a trehalose based dry powder product but it also reconstituted well at and above trehalose/lipid weight ratio of 5.6.
  • the yield of the DPP was excellent and above 75% at and above trehalose/lipid weight ratio of 5.6.
  • the dry powder particle size was higher than 5 micron (81 pm) and the post reconstituted mRNA-LNP particle size was also higher (134 nm) for the dry powder prepared with a lower trehalose/lipid ratio of 2.8. No aggregates were detected in the reconstituted product at and above trehalose/lipid weight ratio of 5.6 as shown in the microscopic images (FIG. 4C).
  • Table 6 shows the dry powder characteristics and post reconstitution composition and characteristics with trehalose as the sugar excipient.
  • FIG. 4A shows the appearances of reconstituted dry powder with different amounts of trehalose as excipient (left: 2.5% trehalose (w/v); middle: 5% trehalose (w/v); right 7% trehalose (w/v)).
  • FIG. 4B shows the appearance of reconstituted dry powder with 1.25% trehalose (w/v) as excipient.
  • FIG. 4C shows no aggregates were observed using a microscope for reconstituted dry powder with 2.5% trehalose (w/v) as excipient as compared to water alone.
  • FIG. 4A shows the appearances of reconstituted dry powder with different amounts of trehalose as excipient (left: 2.5% trehalose (w/v); middle: 5% trehalose (w/v); right 7% trehalose (w/v)).
  • FIG. 4B shows the appearance of reconstituted dry
  • Table 7 shows the dry powder characteristics and post reconstitution composition and characteristics with a low amount of trehalose as the sugar excipient in the preparation of the DPP and reconstituted with different amounts of trehalose added externally as part of the reconstituting solvent.
  • FIG. 5 shows the appearances of reconstituted dry powder with trehalose added as part of the reconstituting solvent at different concentrations.
  • Table 4 Dry powder characteristics and post reconstitution composition and characteristics of mRNA-LNP dry powder with mannitol as excipient.
  • Table 5 Dry powder characteristics and post reconstitution composition and characteristics of mRNA-LNP dry powder with sucrose as excipient.
  • Table 6 Dry powder characteristics and post reconstitution composition and characteristics of mRNA-LNP dry powder with trehalose as excipient.
  • Table 7 Dry powder characteristics and post reconstitution composition and characteristics of mRNA-LNP dry powder with trehalose as excipient and reconstituted with trehalose added externally as part of the reconstituting solvent.
  • This experiment analyzed the physical characteristics of the mRNA-LNP based DPP generated in Example 1.
  • the size of mRNA-LNPs was measured using Zetasizer (Malvern). Pre- and postspray drying encapsulation of the mRNA was determined using standard ribogreen assay. The size of the DPP particles was measured using Mastersizer (Malvern). The mRNA was extracted from the DPP and its integrity was determined using capillary electrophoresis (CE, Agilent). Optical microscope was used to identify the aggregates in the reconstituted dry powder.
  • Example 4 Thermostability of trehalose based mRNA-LNP dry powder product
  • thermostability of the mRNA-LNP based DPP generated using trehalose as the sugar excipient post reconstitution concentration 5% (w/v)
  • PDI poly dispersity index
  • EE encapsulation efficiency
  • mRNA integrity The LNP formulation used in this example contained CKK-E10 as the cationic lipid and the LNPs were loaded with mRNA encoding an influenza antigen.
  • Example 5 In vivo expression of reconstituted mRNA-LNP DPP by intravenous delivery
  • Trehalose DP trehalose dry powder
  • Standard a control mRNA
  • the reconstituted trehalose dry powder has comparable characteristics as a liquid control which contains the same formulation without spray drying.
  • FIG. 8B no significant difference in protein expression was detected between the liquid control and the reconstituted DPP.
  • Table 9 Characteristics of liquid control and reconstituted trehalose dry powder.
  • This Example provides possible uses of mRNA-LNP dry powder, manufactured with sugar or sugar alcohol excipients, for treatment of one or more conditions.
  • the mRNA-LNP DPP will be compressed into various implants such as needle along with other excipients and can be directly injected using devices for different routes of administration such as oral, subcutaneous, and intramuscular.
  • the mRNA-LNP DPP will be administered as powder or blended with excipients to manufacture granules, compressed into tablets that will be used for mucosal delivery via sublingual and buccal routes of administration.
  • the additional excipients can be selected from commonly used compression agents such as microcrystalline cellulose (MCC), microfine cellulose (MFC), directly compressible starch (Sta-Rx 1500), dibasic calcium phosphate (DCP), spray dried lactose (SD lactose), anhydrous lactose (USP), fast Flo® lactose, spray-crystallized maltose and dextrose, crystalline sorbitol, mannitol, sucrose.
  • MCC microcrystalline cellulose
  • MFC microfine cellulose
  • DCP dibasic calcium phosphate
  • SD lactose spray dried lactose
  • USB anhydrous lactose
  • fast Flo® lactose spray-crystallized maltose and dextrose
  • crystalline sorbitol mannitol, sucrose.
  • Some of the commonly used granulating agents are sucrose, acacia, methyl cellulose, liquid glucose, tragacanth, ethyl cellulose, gelatin, hydroxy propyl methyl cellulose (HPMC), starch paste, hydroxy propyl cellulose, pregelatinized starch, sodium carboxy methyl cellulose, alginic acid, polyvinyl pyrrolidone (PVP), cellulose, polyethylene glycol (PEG), polyvinyl alcohols, polymethacrylates.
  • Example 7 In vivo expression of reconstituted mRNA-LNP DPP by intramuscular delivery
  • DPP was prepared by spray drying an mRNA-LNP formulation comprising mRNA encoding human erythropoietin (hEPO) in the presence of trehalose as the sugar excipient.
  • the LNPs used in this example comprise cKK-ElO as the cationic lipid.
  • the DPP was reconstituted and administered intramuscularly in mice at a dosage of 0.1 pg/animal. After 24 hours of administration, the protein expression of the hEPO-coding mRNA was evaluated.
  • Example 8 Improved process for storing mRNA-LNP based DPP
  • This example describes an improved process for storing mRNA-LNP based DPP in a controlled environment.
  • tubes containing DPPs were stored in vacuum-sealed bags, which were then kept in a chamber containing desiccant and filled with nitrogen gas. Desiccant absorbs the moisture in the chamber and nitrogen gas provides an inert overlay, which created a control, low humidity storage condition.
  • Samples of each DPP were removed from the tube and reconstituted for thermostability testing, including change in particle size, PDI, encapsulation efficiency, and mRNA integrity, at different time point.
  • the DPP prepared with mRNA encoding hEPO was also tested for the mRNA expression at different time points.
  • This DPP was prepared by encapsulating the 4 different mRNAs in LNPs containing GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid and spray-dried in the presence of trehalose as the excipient. Samples of the DPP were removed from the tube and reconstituted for thermostability testing, including change in particle size, PDI, encapsulation efficiency, and mRNA integrity, at different time point. [394] As shown in FIG.

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Abstract

La présente invention concerne l'utilisation de particules de poudre sèche d'ARNm reconstituées pour une administration parentérale. La présente invention concerne également un procédé de génération de particules de poudre sèche complétées par des excipients appropriés pour une thermostabilité et une expression in vivo optimales.
PCT/EP2024/060446 2023-04-17 2024-04-17 Formulations de poudre sèche reconstituables et leurs procédés d'utilisation Pending WO2024218166A1 (fr)

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