Lipid nanoparticle formulations, and methods of use thereof

Abstract

The present disclosure provides lipid nanoparticles or lipid nanoparticle formulations. The lipid nanoparticle formulation comprises one or more nucleic acids; a lipid nanoparticle encapsulating one or more nucleic acids; wherein the lipid nanoparticle comprises lipid components, a first buffer solution having a pH in the range of 4 to 6.5, and a stabilizing agent; wherein the stabilizing agent comprises glycerol and the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation. In some embodiments, the lipid nanoparticle formulation further comprises a second buffer solution having a pH in the range of 6 to 8, wherein the second buffer solution dilutes the first buffer solution.

Description

Attorney Reference: RDWD-054WOLIPID NANOPARTICLE FORMULATIONS, AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 718,325, filed November 8, 2024, the disclosure of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present invention relates generally to the field of nucleic acid delivery, and more specifically to lipid nanoparticle (LNP) formulations, methods for making LNP formulations, and methods of using LNP formulations to deliver therapeutic agents.BACKGROUND OF THE INVENTION

[0003] N ucleic acid-based therapeutics find use in various medical applications such as prophylactic vaccines, cancer immunotherapy, or gene editing. To deliver nucleic acids, various drug delivery vehicles have been developed over the years. These vehicles protect the nucleic acids from degradation when delivered in vivo. Lipid nanoparticles (LNPs) have been amongst the most promising vehicles to deliver therapeutic agents. LNPs generally contain four lipids - an ionizable cationic lipid, helper lipid, cholesterol, and a polyethylene glycol (PEG)-lipid conjugate.

[0004] LNPs are commonly used to encapsulate mRNA for efficient in vivo delivery of mRNA. As the major carrier for mRNA-based therapeutics, LNPs have been studied extensively for their delivery performance. Yet, stability of mRNA-LNPs remains challenging for pharmaceutical use, as shown by their stringent handling procedures and deep-freezing conditions (-80 °C) for storage. Over time, increase in size and RNA degradation is usually observed. Though a variety of LNPs have been developed, improvements in storage stability, efficacy, and specificity are still desired.BRIEF SUMMARY OF THE INVENTION

[0005] The present disclosure provides lipid nanoparticles or lipid nanoparticle formulations. The lipid nanoparticle formulation comprises one or more nucleic acids; a lipid nanoparticle encapsulating one or more nucleic acids; wherein the lipid nanoparticle comprises lipidAttorney Reference: RDWD-054WO components, a first buffer solution having a pH in the range of 4 to 6.5, and a stabilizing agent; wherein the stabilizing agent comprises glycerol and the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation. In some embodiments, the lipid nanoparticle formulation further comprises a second buffer solution having a pH in the range of 6 to 8, wherein the second buffer solution dilutes the first buffer solution.

[0006] In some embodiments, the first buffer solution comprises sodium citrate or citric acid, and the second buffer solution comprises tris(hydroxymethyl)aminomethane or its salt form.

[0007] In some embodiments, the lipid nanoparticle formulation upon storage for a period of at least one week at a temperature of about 40° C, exhibits change in the integrity of the nucleic acid by less than about 50%. In some embodiments, the lipid nanoparticle formulation upon storage for a period of at least four weeks at a temperature of about 25° C, exhibits change in the integrity of the nucleic acid by less than about 50%. In some embodiments, the lipid nanoparticle formulation upon storage for a period of at least four months at a temperature of about 5° C, exhibits change in the integrity of the nucleic acid by less than about 50%.

[0008] In some embodiments, the lipid nanoparticle formulation following at least three rounds of freezing at a temperature of about -80° C and thawing, exhibits degradation by less than about 20%. In some embodiments, the lipid nanoparticle formulation following at least three rounds of freezing at a temperature of about -20° C and thawing, exhibits degradation by less than about 20%.

[0009] Also provided herein is the use of the lipid nanoparticle formulation in the manufacture of a medicament for delivering the nucleic acid in vivo, wherein the medicament is administered intravenously, subcutaneously, intramuscularly, or by pulmonary delivery.

[0010] Also provided herein is a pharmaceutical composition comprisingthe lipid nanoparticle formulation described herein and a pharmaceutically acceptable carrier.

[0011] Also provided are methods of making the lipid nanoparticle formulation. The method of making the lipid nanoparticle formulation comprises a) mixing an aqueous solution comprising one or more nucleic acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; e) adding a stabilizing agent in any one or more of the stepsAttorney Reference: RDWD-054WO a) to d); wherein the stabilizing agent connprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0012] In certain embodiments, the method further comprises adding the second buffer solution in step d). In certain embodiments, the method further comprises freezing and storing the lipid nanoparticle formulation.

[0013] Also provided are methods of treating or preventing a disease or disorder by administering to a subject in need thereof the lipid nanoparticle formulation as described herein.

[0014] Also provided is a lipid nanoparticle formulation prepared by a method comprising mixing an aqueous solution comprising one or more nucleic acids, a first buffer solution having a pH in the range of 4 to 6.5 and a stabilizing agent with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A and IB demonstrate the impact of the first buffer solution on LNP stability under freeze / thaw conditions.

[0016] FIGS. 2A and 2B demonstrate the impact of the first buffer solution on LNP thermal stability. FIGS. 2C and 2D depict mRNA integrity and relative mRNA integrity. FIG. 2E illustrates staggered electropherogram of initial mRNA in LNP (TO, black), T3D (blue) and T7D (red) for formulation 2A.

[0017] FIGS. 3A and 3B demonstrate the impact of the second buffer solution and stabilizing excipients (glycerol, DMSO, and sucrose) on LNP thermal response at 40°C. FIGS. 3C and 3D depict mRNA integrity and relative mRNA integrity.

[0018] FIGS. 4A and 4B demonstrate the impact of the second buffer solution on LNP thermal response at 25°C. FIGS. 4C and 4D depict mRNA integrity and relative mRNA integrity.

[0019] FIGS. 5A and 5B demonstrate the impact of the second buffer solution on LNP thermal response at 5°C. FIGS. 5C and 5D depict mRNA integrity and relative mRNA integrity.

[0020] FIG. 6 depicts mRNA stability in different buffers and pH conditions.

[0021] FIGS. 7A and 7B demonstrate the impact of the second buffer solution on LNP freezethaw and thermal response. FIGS. 7C and 7D depict mRNA integrity and relative mRNA integrity.

[0022] FIGS. 8A and 8B demonstrate the effect of additional stabilizing excipients in maintaining LNP stability during freeze / thaw conditions.Attorney Reference: RDWD-054WO

[0023] FIGS. 9A and 9B demonstrate the impact of the different ionizable lipids, N / P ratios and stabilizing excipients on LNP freeze-thaw stability.

[0024] FIG. 10 illustrates the LNP production process.DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure provides lipid nanoparticle formulations, methods for making the lipid nanoparticle formulations, and methods for delivering a therapeutic agent using the lipid nanoparticle formulations.

[0026] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0027] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0029] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It isAttorney Reference: RDWD-054WO further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0030] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent the definition or usage of any term herein conflicts with a definition or usage of a term in an application or reference incorporated by reference herein, the instant application shall control.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.DEFINITIONS

[0032] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0033] As used herein, the term "batch" refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing order during the same cycle of manufacture. A batch may refer to an amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. In some embodiments, a batch would include the mRNA produced from a reaction in which not all reagents and / or components are supplemented and / or replenished as the reaction progresses. The term "not in a single batch" would not mean mRNA synthesized at different times that are combined to achieve the desired amount.Attorney Reference: RDWD-054WO

[0034] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, 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 othertissues (also referred to as "systemic distribution" or "systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.

[0035] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalent, refers to the process of confining an mRNA molecule within a nanoparticle.

[0036] Engineered or mutant: As used herein, the terms "engineered" or " mutant", or grammatical equivalents refer to a nucleotide or protein sequence comprising one or more modifications compared to its naturally-occurring sequence, including but not limited to deletions, insertions of heterologous nucleic acids or amino acids, inversions, substitutions, or combinations thereof.

[0037] Expression: As used herein, "expression" of a nucleic acid sequence 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). In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0038] Functional: As used herein, 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.

[0039] Half-life: As used herein, the term "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.

[0040] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A "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.Attorney Reference: RDWD-054WO

[0041] Impurities: As used herein, the term "impurities" refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.

[0042] In Vitro: As used herein, the term "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 multicellular organism.

[0043] In Vivo: As used herein, the term "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).

[0044] Isolated: As used herein, the term "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. In some embodiments, 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. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0045] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.Attorney Reference: RDWD-054WO

[0046] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl- cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages). In some embodiments, the present invention is specifically directed to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order toAttorney Reference: RDWD-054WO facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0047] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.

[0048] Pharmaceutically acceptable: The term "pharmaceutically acceptable" as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0049] Stable: As used herein, the term "stable" protein or its grammatical equivalents refer to protein that retains its physical stability and / or biological activity. In one embodiment, protein stability is determined based on the percentage of monomer protein in the solution, at a low percentage of degraded (e.g., fragmented) and / or aggregated protein. In one embodiment, a stable engineered protein retains or exhibits an enhanced half-life as compared to a wild-type protein. In one embodiment, a stable engineered protein is less prone to ubiquitination that leads to proteolysis as compared to a wild-type protein.

[0050] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, 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. The term "subject" is used herein interchangeably with "individual" or "patient." 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.

[0051] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.Attorney Reference: RDWD-054WO

[0052] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.LIPID NANOPARTICLE FORMULATIONS

[0053] P rovided by the present disclosure are lipid nanoparticle formulations. The lipid nanoparticle formulation includes one or more nucleic acids; a lipid nanoparticle encapsulating one or more nucleic acids; where the lipid nanoparticle includes lipid components, a first buffer solution having a pH in the range of 4 to 6.5, and a stabilizing agent; where the stabilizing agent comprises glycerol and the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0054] The lipid nanoparticle formulation may comprise any type of nucleic acid. A nucleic acid may be selected from the group comprising (but not limited to) DNA, messenger RNA (mRNA), self-amplifying RNA (saRNA), small nuclear RNA (snRNA), circular RNA, guide RNA (gRNA), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro RNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). In some embodiments, the lipid nanoparticle formulation may comprise one or more nucleic acids.

[0055] The lipid nanoparticle formulation may comprise any mRNA. mRNA is typically thought of as the type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotic organisms, mRNA processing comprises the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. A typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. The presence of the cap may provide resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. Messenger RNA typically is translated by the ribosomes into a series of amino acids that make up a protein or a peptide.

[0056] Any mRNA capable of being translated into one or more peptides (e.g., antigens) or peptide fragments is contemplated as within the scope of the present invention. In someAttorney Reference: RDWD-054WO embodiments, an mRNA encodes one or more naturally occurring peptides. In some embodiments, an mRNA encodes one or more modified or non-natural peptides.

[0057] In some embodiments an mRNA encodes an intracellular protein or peptide. In some embodiments, an mRNA encodes a cytosolic protein or peptide. In some embodiments, an mRNA encodes a protein or peptide associated with the actin cytoskeleton. In some embodiments, an mRNA encodes a protein or peptide associated with the plasma membrane. In some specific embodiments, an mRNA encodes a transmembrane protein or peptide. In some specific embodiments an mRNA encodes an ion channel protein or peptide. In some embodiments, an mRNA encodes a perinuclear protein or peptide. In some embodiments, an mRNA encodes a nuclear protein or peptide. In some specific embodiments, an mRNA encodes a transcription factor. In some embodiments, an mRNA encodes a chaperone protein or peptide. In some embodiments, an mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzyme associated with urea cycle or lysosomal storage metabolic disorders). In some embodiments, an mRNA encodes a protein or peptide involved in cellular metabolism, DNA repair, transcription and / or translation. In some embodiments, an mRNA encodes an extracellular protein or peptide. In some embodiments, an mRNA encodes a protein or peptide associated with the extracellular matrix. In some embodiments an mRNA encodes a secreted protein or peptide. In specific embodiments, an mRNA used in the composition and methods of the invention may be used to express functional proteins or enzymes that are excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and / or neurotransmitters).

[0058] In some embodiments, the lipid nanoparticle formulation may comprise one or more nucleic acids to be encapsulated at various concentrations. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration from about 0.01 mg / mL to about 2 mg / mL. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration ranging from about 0.01-2.0 mg / mL, 0.01-1.9 mg / mL, 0.01-1.8 mg / mL, 0.01-1.7 mg / mL, 0.01-1.6 mg / mL, 0.01-1.5 mg / mL, 0.01-1.4 mg / mL, 0.01-1.3 mg / mL, 0.01-1.2 mg / mL, 0.01-1.1 mg / mL, 0.01-1.0 mg / mL, 0.01-0.9 mg / mL, 0.01-0.8 mg / mL, 0.01-0.7 mg / mL, 0.01-0.6 mg / mL, 0.01-0.5 mg / mL, 0.01-0.4 mg / mL, 0.01-0.3 mg / mL, 0.01-0.2 mg / mL, or 0.01-0.1 mg / mL.

[0059] In certain embodiments, the lipid nanoparticle formulation may comprise lipid nanoparticle encapsulating one or more nucleic acids. In some embodiments, the lipidAttorney Reference: RDWD-054WO nanoparticle comprises lipid components. The lipid components may be a combination of one or more cationic lipids and non-cationic lipids.

[0060] As used herein, the phrase "non-cationic lipid" or "neutral lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non- cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2- oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids.

[0061] Cationic lipids include, but are not limited to, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 5-carboxyspermylglycinedioctadecylamide (DOGS), 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), 1,2- Dioleoyl-3-Dimethylammonium-Propane (DODAP), l,2-Dioleoyl-3-Trimethylammonium- Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy- N,N-dimethyl-3-aminopropane (DODMA), l,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 bromide (DMRIE), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane or (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolaneAttorney Reference: RDWD-054WO(DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA), [(4- Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), (6Z,9Z,28Z,31Z)- Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2-[2,2- bis[(9Z,12Z)-octadeca-9,12-dienyl]-l,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2- DMA), 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102), or mixtures thereof.

[0062] In some embodiments, the ionizable cationic lipid is present in a molar percentage of the lipid nanoparticle from about 30% to about 60%. In some embodiments, the molar percentage of the ionizable cationic lipid is present from about 35% to about 55%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%.

[0063] In some embodiments, the lipid components may comprise a phospholipid. Phospholipids include, but are not limited to l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (1,2- DSPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (1,2-DSPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-DOPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1,2-POPC), l-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (1,2-SOPC), or mixtures thereof.

[0064] In some embodiments, the phospholipid is present in a molar percentage of the lipid nanoparticle from about 0% to about 30%. In some embodiments, the molar percentage of the phospholipid is present from about 5% to about 25%. In some embodiments, the molar percentage of the phospholipid is about 0%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%.

[0065] In some embodiments, the lipid components may comprise a sterol-based lipid. Sterol- based lipids include, but are not limited to cholesterol, cholesterol sulfate, campesterol, betasitosterol, stigamasterol, or mixtures thereof.

[0066] In some embodiments, the sterol-based lipid is present in a molar percentage of the lipid nanoparticle from about 15% to about 50%. In some embodiments, the molar percentage of the sterol-based lipid is present from about 20% to about 45%. In some embodiments, the molar percentage of the sterol-based lipid is about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%.

[0067] In some embodiments, the lipid components may comprise a PEGylated lipid. PEGylated lipids include, but are not limited to PEG-modified phosphatidylethanolamine, PEG-Attorney Reference: RDWD-054WO modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, DSPE- PEG(2000), DSG-PEG (2000), DPG-PEG (2000), ALC-0159, DMG-PEG(2000) or mixtures thereof.

[0068] In some embodiments, the PEGylated lipid is present in a molar percentage of the lipid nanoparticle from about 0.01% to about 10%. In some embodiments, the molar percentage of the PEGylated lipid is present from about 1% to about 10%. In some embodiments, the molar percentage of the PEGylated lipid is about 0.01%, or about 0.5%, or about 1%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%. In some embodiments, the PEGylated lipid comprises a PEG component from about 1000 to about 10,000 Daltons.

[0069] In some cases, the lipid components are selected from a group consisting of an ionizable cationic lipid, phospholipid, a PEGylated lipid and a sterol-based lipid. In some embodiments, the lipid nanoparticle comprises about 30-60 mol % ionizable cationic lipid; about 0-30 mol % phospholipid; about 15-50 mol % sterol-based lipid; and about 0.01-10 mol % PEGylated lipid. The selection of sterol-based cationic lipids, non-cationic lipids and / or PEGylated lipids which comprise the lipid components 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.

[0070] The lipid nanoparticle may have a diameter (mean particle diameter) from about 15 nm to about 300 nm. In some embodiments, the lipid nanoparticle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In some embodiments, the lipid nanoparticle has a diameter from about 15 nm to about 100 nm. In some embodiments, the lipid nanoparticle has a diameter from about 15 nm to about 50 nm. In some embodiments, the lipid nanoparticle has a diameter from about 15 to about 20 nm.

[0071] Suitable stabilizing agent for the formulations and methods disclosed herein includes a cryoprotectant. For example, the cryoprotectant comprises one or more cryoprotective agents, and each of the one or more cryoprotective agents is independently a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4- pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine (e.g., NDSB-201 (3-(l-pyridino)-l-propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethyleneAttorney Reference: RDWD-054WO glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)- trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof) or any combination thereof.

[0072] In some embodiments, the formulation also includes one or more salts. For example, the formulation includes one or more salts selected from the group consisting of lithium salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, or any hydrate thereof), magnesium salts (e.g., magnesium acetate or a hydrate thereof), and sodium salts (e.g., sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof). For another example, the formulation comprises one or more sodium salts. For yet another example, the formulation comprises sodium chloride. In some embodiments, the formulation can be free of one or more salts.

[0073] In some embodiments, the stabilizing agent comprises glycerol. The concentration of the stabilizing agent ranges from about 0.1% to about 30% by weight (for example, from about 0.1% to about 25% by weight, from about 0.5% to about 25% by weight, from about 1% to about 20% by weight or from about 5% to about 15% by weight).

[0074] In some embodiments, the lipid nanoparticle comprises a first buffer solution having a pH in the range of 4 to 6.5. In some embodiments, the first buffer solution may have a pH ranging from about 4 to about 6.5, about 4.5 to about 6.5, about 4.8 to about 6.5, about 5.0 to about 6.5, about 5.5 to about 6.5, about 5.8 to about 6.5, about 6.0 to about 6.5. In some embodiments, a suitable first buffer solution may have a pH of or no greater than 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5.

[0075] The first buffer solution includes, but is not limited to, sodium citrate, potassium citrate, calcium citrate, citric acid or a combination thereof. In some embodiments, the first buffer solution is sodium citrate. In some embodiments, the first buffer solution is citric acid. In some embodiments, the first buffer solution is at a concentration ranging from about 5-100 mM,Attorney Reference: RDWD-054WO from about 5-90 mM, from about 10-80 mM, from about 15-70 mM, from about 20-60 mM. In some embodiments, the first buffer solution is at a concentration of orgreaterthan about 5 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

[0076] In some embodiments, the lipid nanoparticle further comprises a second buffer solution having a pH in the range of 6 to 8. In some embodiments, the second buffer solution may have a pH ranging from about 6 to about 8, about 6.5 to about 8, about 6.8 to about 8, about 7.0 to about 8, about 7.5 to about 8, about 7.8 to about 8. In some embodiments, a suitable second buffer solution may have a pH of or no greater than 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0.

[0077] The second buffer solution includes, but is not limited to, tris(hydroxymethyl)aminomethane or its salt form. In some embodiments, the second buffer solution is tris(hydroxymethyl)aminomethane. In some embodiments, the second buffer solution is phosphate. In some embodiments, the second buffer solution is at a concentration ranging from about 5-100 mM, from about 5-90 mM, from about 10-80 mM, from about 15-70 mM, from about 20-60 mM. In some embodiments, the second buffer solution is at a concentration of or greater than about 5 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

[0078] In some embodiments, the second buffer solution dilutes the first buffer solution. The second buffer solution displaces the first buffer solution in the lipid nanoparticle.

[0079] In some embodiments, upon storage of the lipid nanoparticle formulation for a period of at least one week at a temperature of about 40° C, the integrity of the nucleic acid changes by less than about 50%. In some embodiments, the integrity of the nucleic acid encapsulated by the lipid nanoparticle in the lipid nanoparticle formulation when stored for at least one week at a temperature of about 40° C, changes by about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less as compared to the lipid nanoparticle formulation produced by a conventional method.

[0080] In some embodiments, upon storage of the lipid nanoparticle formulation for a period of at least four weeks at a temperature of about 25° C, the integrity of the nucleic acid changes by less than about 50%. In some embodiments, the integrity of the nucleic acid encapsulated by the lipid nanoparticle in the lipid nanoparticle formulation when stored for at least four weeks at a temperature of about 25° C, changes by about 50% or less, about 40% or less, about 30% orAttorney Reference: RDWD-054WO less, about 20% or less, or about 10% or less as compared to the lipid nanoparticle formulation produced by a conventional method.

[0081] In some embodiments, upon storage of the lipid nanoparticle formulation for a period of at least four months at a temperature of about 5° C, the integrity of the nucleic acid changes by less than about 50%. In some embodiments, the integrity of the nucleic acid encapsulated by the lipid nanoparticle in the lipid nanoparticle formulation when stored for at least four months at a temperature of about 5° C, changes by about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less as compared to the lipid nanoparticle formulation produced by a conventional method.

[0082] In some embodiments, following at least three rounds of freezing at a temperature of about -80° Cand thawing, the lipid nanoparticle formulation exhibits degradation less than about 20%. In some embodiments, the lipid nanoparticle formulation following at least three rounds of freezing at a temperature of about -80° C and thawing, exhibits degradation by about 20% or less, or about 15% or less or about 10% or less as compared to the lipid nanoparticle formulation produced by a conventional method.

[0083] In some embodiments, following at least three rounds of freezing at a temperature of about -20° C and thawing, the lipid nanoparticle formulation exhibits degradation of less than about 20%. In some embodiments, the lipid nanoparticle formulation following at least three rounds of freezing at a temperature of about -20° C and thawing, exhibits degradation by about 20% or less, or about 15% or less or about 10% or less as compared to the lipid nanoparticle formulation produced by a conventional method.

[0084] In some embodiments, the lipid nanoparticle formulation may be used of in the manufacture of a medicament for delivering the nucleic acid in vivo, wherein the medicament is administered intravenously, subcutaneously, intramuscularly, or by pulmonary delivery. In some embodiments, the nucleic acid is selected from mRNA, saRNA (self-amplifying), circular RNA, DNA, and / or siRNA, miRNA.PHARMACEUTICAL COMPOSITIONS

[0085] Provided are pharmaceutical compositions comprising the lipid nanoparticle formulations. The pharmaceutical composition comprises the lipid nanoparticle formulation and a pharmaceutically acceptable carrier. Representative pharmaceutically acceptable carriers orAttorney Reference: RDWD-054WO diluents include solutions for intravenous injection (e.g., saline or dextrose). The formulation can take the form of a cream, ointment, gel, suspension, or emulsion.

[0086] In some embodiments, the pharmaceutical compositions are formulated for administration: orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion. In some embodiments, the pharmaceutical compositions are formulated for intravenous or intraarterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical compositions are formulated as a unit dose.METHODS

[0087] Provided are methods for making the lipid nanoparticle formulation. The method comprises a) mixing an aqueous solution comprising one or more nucleic acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; e) adding a stabilizing agent in any one or more of the steps a) to d); wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0088] FIG. 10 describes a diagram of the lipid nanoparticle production process. Particularly, potential addition points of stabilization agent and buffer solutions are shown. For example, stabilizing agent can be added in step a), step b), step c) or step d). The second buffer solution can be added step b), step c) or step d).Attorney Reference: RDWD-054WO

[0089] In one of the embodiments, the method comprises adding stabilizing agent in step a). For example, the method comprises a) mixing an aqueous solution comprising one or more nucleic acids, a first buffer solution having a pH in the range of 4 to 6.5 and a stabilizing agent with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0090] In another embodiment, the method comprises adding stabilizing agent in step b). For example, the method comprises a) mixing an aqueous solution comprising one or more nucleic acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution, and adding a stabilizing agent; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0091] In another embodiment, the method comprises adding stabilizing agent in step c). For example, the method comprises a) mixing an aqueous solution comprising one or more nucleic acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution, and adding a stabilizing agent; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0092] In another embodiment, the method comprises adding stabilizing agent in step d). For example, the method comprises a) mixing an aqueous solution comprising one or more nucleicAttorney Reference: RDWD-054WO acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution, and adding a stabilizing agent to form the lipid nanoparticle formulation; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0093] In some embodiments, the method further comprises adding the second buffer solution in step (d).

[0094] In some embodiments, the methods of the present disclosure provide an aqueous solution comprising one or more nucleic acids, a first buffer solution having a pH in the range of 4 to 6.5 and a stabilizing agent. The aqueous solution may be provided to be mixed or added to a solution comprising lipid components to form a lipid nanoparticle solution such that the nucleic acid may be encapsulated in the lipid nanoparticle.

[0095] N ucleic acid is as described in the above paragraphs. In some embodiments, the nucleic acid is an RNA (e.g., mRNA). In some embodiments, the lipid nanoparticle formulation may comprise one or more nucleic acids to be encapsulated at various concentrations. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration from about 0.01 mg / mL to about 2 mg / mL. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration ranging from about 0.01-2.0 mg / mL, 0.01-1.9 mg / mL, 0.01-1.8 mg / mL, 0.01-1.7 mg / mL, 0.01-1.6 mg / mL, 0.01-1.5 mg / mL, 0.01-1.4 mg / mL, 0.01-1.3 mg / mL, 0.01-1.2 mg / mL, 0.01-1.1 mg / mL, 0.01-1.0 mg / mL, 0.01-0.9 mg / mL, 0.01-0.8 mg / mL, 0.01-0.7 mg / mL, 0.01-0.6 mg / mL, 0.01-0.5 mg / mL, 0.01-0.4 mg / mL, 0.01-0.3 mg / mL, 0.01-0.2 mg / mL, or 0.01-0.1 mg / mL.

[0096] The lipid components are as described in the above paragraphs. In some embodiments, the lipid components are selected from a group consisting of an ionizable cationic lipid, phospholipid, a PEGylated lipid and a sterol-based lipid. Suitable ionizable cationic lipids, phospholipids, PEGylated lipids and sterol-based lipids for the methods are described in the above paragraphs. In some embodiments, the ionizable cationic lipid is selected from a group consisting of DODAP, DODMA, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA and SM-102. In some embodiments, the phospholipid is selected from a group consisting of 1,2-DOPE, 1,2-DSPE, 1,2-Attorney Reference: RDWD-054WODOPC, 1,2-POPC, 1,2-SOPC, and DSPC. In some embodiments, the PEGylated lipid is selected from a group consisting of DSPE-PEG(2000), DSG-PEG (2000), DPG-PEG (2000), ALC-0159 and DMG-PEG(2000). In some embodiments, the sterol-based lipid is selected from a group consisting of cholesterol, cholesterol sulfate, campesterol, beta-sitosterol, stigamasterol. In some embodiments, the lipid nanoparticle comprises about 30-60 mol % ionizable cationic lipid; about 0-30 mol % phospholipid; about 15-50 mol % sterol-based lipid; and about 0.01-10 mol % PEGylated lipid.

[0097] The stabilizing agent is as described in the above paragraphs. In some embodiments, the stabilizing agent comprises glycerol. The concentration of the stabilizing agent ranges from about 0.1% to about 30% by weight (for example, from about 0.1% to about 25% by weight, from about 0.5% to about 25% by weight, from about 1% to about 20% by weight or from about 5% to about 15% by weight).

[0098] The first buffer solution is as described in the above paragraphs. In some embodiments, the first buffer solution has a pH in the range of 4 to 6.5. In some embodiments, the first buffer solution may have a pH ranging from about 4 to about 6.5, about 4.5 to about 6.5, about 4.8 to about 6.5, about 5.0 to about 6.5, about 5.5 to about 6.5, about 5.8 to about 6.5, about 6.0 to about 6.5. In some embodiments, the first buffer solution is sodium citrate. In some embodiments, the first buffer solution is citric acid.

[0099] In some embodiments, the method comprises mixing an aqueous solution and a solution comprising lipid components to form a lipid nanoparticle solution. In some embodiments, the mixing step is performed with a T-junction, confined impinging jets, microfluidic mixer, vortex mixer, co-axial mixer, or crossflow mixer.

[0100] In some embodiments, the method further comprises exchanging the first buffer solution with a second buffer solution, wherein the second buffer solution dilutes and displaces the first buffer solution. The second buffer solution is as described in the above paragraphs. In some embodiments, the second buffer solution has a pH in the range of 6 to 8. In some embodiments, the second buffer solution may have a pH ranging from about 6 to about 8, about 6.5 to about 8, about 6.8 to about 8, about 7.0 to about 8, about 7.5 to about 8, about 7.8 to about 8. In some embodiments, a suitable second buffer solution may have a pH of or no greater than 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0.

[0101] This step of exchanging buffer solutions may result in purification of the lipid nanoparticles. Various other purification methods may also be used. In some embodiments, lipidAttorney Reference: RDWD-054WO nanoparticles are purified using dialysis or Tangential Flow Filtration. Tangential flow filtration (TFF), also referred to as cross-flow filtration, is a type of filtration wherein the material to be filtered is passed tangentially across a filter rather than through it. In TFF, undesired permeate passes through the filter, while the desired retentate passes along the filter and is collected downstream. It is important to note that the desired material is typically contained in the retentate in TFF. Depending upon the material to be filtered, TFF is usually used for either microfiltration or ultrafiltration. Microfiltration is typically defined as instances where the filter has a pore size of between 0.05 pm and 1.0 pm, inclusive, while ultrafiltration typically involves filters with a pore size of less than 0.05 pm. Pore size also determines the nominal molecular weight limits (NMWL), also referred to as the molecular weight cut off (MWCO) for a particular filter, with microfiltration membranes typically having NMWLs of greater than 1,000 kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1 kDa and 1,000 kDa.

[0102] In some embodiments, the method further comprises freezing the lipid nanoparticle formulation. In some embodiments, the method further comprises storing the lipid nanoparticle formulation. In some embodiments, the method may comprise packing the lipid nanoparticle formulation. As used herein, "packing" refers to storing the lipid nanoparticle formulation in its final state or in-process storage of the lipid nanoparticle formulation before they are placed into final packaging. Modes of storage and / or packing include, but are not limited to, refrigeration in sterile bags, refrigerated or frozen formulations in vials, lyophilized formulations in vials and syringes, etc.

[0103] In some embodiments, the step of storing the lipid nanoparticle formulation comprises lyophilizing the lipid nanoparticle formulation, storing the lyophilized lipid nanoparticle formulation. In some embodiments, cryoprotectant is added to the lipid nanoparticle formulation prior to the lyophilization.

[0104] In some embodiment, the lipid nanoparticle formulation or the lyophilized lipid nanoparticle formulation is stored at a temperature of about -80° C., about -78° C., about -76° C., about -74° C., about -72° C., about -70° C., about -65° C., about -60° C., about -55° C., about -50° C., about -45° C., about -40° C., about -35° C., about -30° C., about -25° C., or about -20° C.

[0105] In some embodiment, the lipid nanoparticle formulation or the lyophilized lipid nanoparticle formulation is stored at a temperature of about -40° C., about -35° C., about -30°Attorney Reference: RDWD-054WOC., about -25° C., about -20° C., about -15° C., about -10° C., about -5° C., about 0° C., about 5° C., about 10° C., about 15° C., about 20° C., or about 25° C..

[0106] Also provided is a method of treating or preventing a disease or disorder. The method comprises administering to a subject in need thereof the lipid nanoparticle formulation as described herein. In some embodiments, the method comprises, administration of the lipid nanoparticle formulation orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, parenterally, rectally, subconjunctival, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via localized perfusion.

[0107] Also provided is a lipid nanoparticle formulation prepared by a method as described herein. The method comprises mixing an aqueous solution comprising one or more nucleic acids, a first buffer solution having a pH in the range of 4 to 6.5 and a stabilizing agent with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

[0108] N ucleic acid is as described in the above paragraphs. In some embodiments, the nucleic acid is an RNA (e.g., mRNA). In some embodiments, the lipid nanoparticle formulation may comprise one or more nucleic acids to be encapsulated at various concentrations. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration from about 0.01 mg / mL to about 2 mg / mL. In some embodiments, the lipid nanoparticle formulation may comprise a nucleic acid at a concentration ranging from about 0.01-2.0 mg / mL, 0.01-1.9 mg / mL, 0.01-1.8 mg / mL, 0.01-1.7 mg / mL, 0.01-1.6 mg / mL, 0.01-1.5 mg / mL, 0.01-1.4 mg / mL, 0.01-1.3 mg / mL, 0.01-1.2 mg / mL, 0.01-1.1 mg / mL, 0.01-1.0 mg / mL, 0.01-0.9 mg / mL, 0.01-0.8 mg / mL, 0.01-0.7 mg / mL, 0.01-0.6 mg / mL, 0.01-0.5 mg / mL, 0.01-0.4 mg / mL, 0.01-0.3 mg / mL, 0.01-0.2 mg / mL, or 0.01-0.1 mg / mL.

[0109] The lipid components are as described in the above paragraphs. In some embodiments, the lipid components are selected from a group consisting of an ionizable cationic lipid,Attorney Reference: RDWD-054WO phospholipid, a PEGylated lipid and a sterol-based lipid. Suitable ionizable cationic lipids, phospholipids, PEGylated lipids and sterol-based lipids for the methods are described in the above paragraphs. In some embodiments, the ionizable cationic lipid is selected from a group consisting of DODAP, DODMA, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA and SM-102. In some embodiments, the phospholipid is selected from a group consisting of 1,2-DOPE, 1,2-DSPE, 1,2- DOPC, 1,2-POPC, 1,2-SOPC, and DSPC. In some embodiments, the PEGylated lipid is selected from a group consisting of DSPE-PEG(2000), DSG-PEG (2000), DPG-PEG (2000), ALC-0159 and DMG-PEG(2000). In some embodiments, the sterol-based lipid is selected from a group consisting of cholesterol, cholesterol sulfate, campesterol, beta-sitosterol, stigamasterol. In some embodiments, the lipid nanoparticle comprises about 30-60 mol % ionizable cationic lipid; about 0-30 mol % phospholipid; about 15-50 mol % sterol-based lipid; and about 0.01-10 mol % PEGylated lipid.

[0110] The stabilizing agent is as described in the above paragraphs. In some embodiments, the stabilizing agent comprises glycerol. The concentration of the stabilizing agent ranges from about 0.1% to about 30% by weight (for example, from about 0.1% to about 25% by weight, from about 0.5% to about 25% by weight, from about 1% to about 20% by weight or from about 5% to about 15% by weight).

[0111] The first buffer solution is as described in the above paragraphs. In some embodiments, the first buffer solution has a pH in the range of 4 to 6.5. In some embodiments, the first buffer solution may have a pH ranging from about 4 to about 6.5, about 4.5 to about 6.5, about 4.8 to about 6.5, about 5.0 to about 6.5, about 5.5 to about 6.5, about 5.8 to about 6.5, about 6.0 to about 6.5. In some embodiments, the first buffer solution is sodium citrate. In some embodiments, the first buffer solution is citric acid.

[0112] In some embodiments, the method further comprises exchanging the first buffer solution with a second buffer solution, wherein the second buffer solution dilutes and displaces the first buffer solution. The second buffer solution is as described in the above paragraphs. In some embodiments, the second buffer solution has a pH in the range of 6 to 8. In some embodiments, the second buffer solution may have a pH ranging from about 6 to about 8, about 6.5 to about 8, about 6.8 to about 8, about 7.0 to about 8, about 7.5 to about 8, about 7.8 to about 8. In some embodiments, a suitable second buffer solution may have a pH of or no greater than 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0.Attorney Reference: RDWD-054WO

[0113] The lipid nanoparticle formulations of the invention can be furtherdistinguished by the encapsulation efficiency. The lipid nanoparticle formulations of the invention are prepared by a method by which nearly 100% of the nucleic acid in the final formation is encapsulated in the particles. In one embodiment, the lipid nanoparticle formulations are prepared by a method by which from about 90 to about 95% of the nucleic acid in the final formation is encapsulated in the particles. In another embodiment, the lipid nanoparticle formulations are prepared by a method by which from about 80 to about 90% of the nucleic acid in the final formation is encapsulated in the particles.EXAMPLES

[0114] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0115] Example 1: Production of lipid nanoparticle formulation

[0116] In order to investigate integrity and storage stability of lipid nanoparticle formulations for use in the delivery of nucleic acids to cells, a range of formulations were prepared and tested. Specifically, the particular elements and ratios thereof in the lipid component of lipid nanoparticles are optimized. Lipid nanoparticles can be made with mixing processes such as microfluidics and T-junction mixing of two solutions, one of which contains the nucleic acids, and first buffer solution, and the other which contains the lipid components. A solution was prepared by combining MC3 or ALC (an ionizable lipid), DSPC (a phospholipid), cholesterol (a structural lipid) and PEG (a PEG lipid) at molar ratios of about 50:10:38.5:1.5. An aqueous solution was prepared by combining mRNA, and sodium citrate (a first buffer solution). Both solutions were mixed to form the lipid nanoparticle solution wherein the lipid nanoparticle solution contains one or more nucleic acids encapsulated by the lipid nanoparticle. The lipid nanoparticle solution is then diluted by adding a second buffer solution, and sodium citrate (first buffer solution) is exchanged with the second buffer solution. The second buffer solution can be a Tris buffer or a phosphate buffer. Following this, the lipid nanoparticle solution is compounded and sterilized and a stabilizing agent is also added, e.g., glycerol or sucrose. As such, the lipid nanoparticle formulations are formed as shown in Table 1 below.Attorney Reference: RDWD-054WO

[0117] Table 1:

[0118] Example 2: Stability of lipid nanoparticle formulation

[0119] Formulations with different first buffer solutions were prepared using MC3 as an ionizable lipid. LNP samples with mRNA as cargo were frozen at -80°C overnight followed by thawing at 2-8°C for 4-6 hours. Samples were analyzed following three (3XFT) F / T cycles. Sodium citrate performed better compared to the other first buffer solutions as shown by the smaller change to LNP diameter (FIG. 1A) and percent of encapsulation (FIG. IB). FIG. 1 indicates the impact of the first buffer solution on LNP freeze-thaw response. LNP samples with different first buffer and pH were frozen at -80°C overnight followed by thawing at 2-8°Cfor4-6 hours. Samples were analyzed following one (1XFT), three (3XFT) and five (5XFT) freeze / thaw cycles. Sodium citrate (SC) showed better performance under F / T conditions in maintaining LNP stabilityAttorney Reference: RDWD-054WO compared to other buffers, as shown by the smallest change to LNP diameter (FIG. 1A) and percent of encapsulation (FIG. IB), this is most obvious when no sucrose is present. SC: sodium citrate; SA: sodium acetate; His: histidine-HCl.

[0120] Formulation conditions impact mRNA stability under thermal stress (FIGS. 2-3). LNP samples with different first buffer solution and pH were under thermal stress conditions at 40°C for one day (T1D), three days (T3D), seven days (T7D) or 14 days (T14D). mRNA purity was analyzed using capillary electrophoresis (CE). Under thermal conditions, sodium citrate (pH 5) had better stability compared to other formulations, based on relative mRNA integrity calculated from the percent of main peak (FIG. 2). LNP physical properties such as LNP size (FIG. 2A) and percentage of encapsulation (FIG. 2B) had minimal change. mRNA in LNP were chemically unstable and showed different degradation rate in different buffers, as shown by the decrease of mRNA integrity derived from the percent of main peak (FIG. 2C). Relative mRNA integrity calculated from the percentage of main peak relative to time zero (TO) is shown in FIG. 2D.

[0121] Detailed profile of mRNA integrity is shown in FIG. 2E with staggered electropherogram of initial mRNA in LNP TO, T3D and T7D for sodium citrate (pH 5). FIG. 2E shows staggered electropherogram of initial mRNA in LNP (TO, black), T3D (blue) and T7D (red) for formulation 2A.

[0122] Relative mRNA integrity at each time point is summarized in below table 2 within the electropherogram.

[0123] Table 2 indicates calculated reaction rate constant for mRNA inside LNP when assembled under different conditions. Run 1 and run 2 are replicate experiments. Reaction rate constants are calculated based on first order kinetics. Sodium citrate (pH 5) is shown to be the condition with the most mRNA stability.

[0124] Table 2:Attorney Reference: RDWD-054WO

[0125] Among all conditions, Sodium citrate at pH 5 (sample 2B) performed best for thermal stress. This is not anticipated from the experiment in FIG. 6. Such outcome is not anticipated from other buffer / pH (his and acetate).

[0126] Example 3: mRNA and lipid nanoparticle stability in second buffer solution and stabilizing excipient

[0127] Another set of lipid nanoparticle formulations were prepared and tested for mRNA stability. Table 3 below enlists the different lipid nanoparticle formulations.

[0128] Table 3:Attorney Reference: RDWD-054WO

[0129] FIG. 3-5 illustrates mRNA stability results of different lipid nanoparticles formulations described in Table 3 above. FIG. 3 depicts the impact of the second buffer solution and stabilizing excipients (glycerol, DMSO, and sucrose) on LNP thermal response at 40°C. LNP samples underwent thermal stress at 40°C for 1 day (ID), 3 days (3D) and 7 days (7D). SP: Sodium Phosphate. LNP physical properties such as LNP size (FIG. 3A) and percentage of encapsulation (FIG. 3B) had minimal change. mRNA in LNP were chemically unstable and showed different degradation rate in different buffers, as shown by the decrease of mRNA integrity derived from the percent of main peak (FIG. 3C). Relative mRNA integrity calculated from the percentage of main peak relative to time zero (TO) was also shown (FIG. 3D). Sample 4B with 10% glycerol showed the best performance in maintaining mRNA integrity. The ability of glycerol to preserve mRNA integrity in LNPs under thermal stress was not expected.

[0130] FIG. 4 demonstrates the impact of the second buffer solution on LNP thermal response at 25°C. LNP samples underwent thermal stress at 25°C for 1 week (ID), 2 weeks (2W), 1 month (IM), 2.5 months (2.5M), and 4 months (4M). SP: Sodium Phosphate. LNP physical properties such as LNP size (FIG. 4A) and percentage of encapsulation (FIG. 4B) had minimal change. mRNA in LNP were chemically unstable and showed different degradation rate in different buffers, as shown by the decrease of mRNA integrity derived from the percent of main peak (FIG. 4C). Relative mRNA integrity calculated from the percentage of main peak relative to time zero (TO) was also shown (FIG. 4D). Sample 4B with 10% glycerol showed the best performance in maintaining mRNA integrity. Ability of glycerol to preserve mRNA integrity in LNPs under accelerated thermal test (25°C) was not expected.Attorney Reference: RDWD-054WO

[0131] FIG. 5 depicts the impact of the second buffer solution on LNP thermal response at 5°C. SP: Sodium Phosphate. LNP samples underwent thermal stress at 5°C for 1 month (IM), 2.5 months (2.5M), 4 months (4M), 6.5 months (6.5M) and 12 months (12M). LNP physical properties such as LNP size (FIG. 5A) and percent of encapsulation (FIG. 5B) had minimal change in most samples. mRNA in LNP were chemically unstable and showed different degradation rate in different buffers, as shown by the decrease of mRNA integrity derived from the percent of main peak (FIG. 5C). Relative mRNA integrity calculated from the percentage of main peak relative to time zero (TO) was also shown (FIG. 5D). Sample 4B with 10% glycerol showed the best performance in maintaining mRNA integrity. The ability of glycerol to preserve mRNA integrity in LNPs under real time 5°C was not expected.

[0132] Table 4 indicates calculated reaction rate constants for mRNA inside LNP when assembled under different conditions. Reaction rates are calculated based on first order kinetics for 40°C, 25°C, 5°C, and -20°C. Sample 4B is shown to be the condition with the most mRNA stability.

[0133] Table 4:

[0134] Example 4: mRNA stability in different buffer solutions and pH conditions

[0135] Another set of formulations were prepared and tested for mRNA stability. Table 5 below enlists the mRNA formulations.

[0136] Table 5 describes formulations with different buffers and pH conditions. Fig. 6 demonstrates mRNA stability in different buffers and pH conditions. The outcome of degradation depends strongly on both pH and buffer and is not predictable, as different buffers at same pHAttorney Reference: RDWD-054WO can have drastically different outcomes. Among all buffers, sodium citrate pH 6, sodium phosphate pH 7 , and tris buffer pH 7.5 performed the best in stabilizing mRNA. The presence ofEDTA reduced degradation rate.

[0137] Table 5:

[0138] Example 5: Impact of second buffer solutions and stabilizing agents on mRNA and lipid nanoparticle stability

[0139] Another set of lipid nanoparticle formulations were prepared and tested for mRNA stability. Table 6 below enlists the different lipid nanoparticle formulations.Attorney Reference: RDWD-054WO samples with different second buffer solutions and pH were frozen at -80°C or -20°C overnight followed by thawing at 2-8°C for 4-6 hours. Samples were analyzed following one (1XFT) and three (3XFT) freeze-thaw cycles. For thermal stress, LNP samples underwent thermal stress at 40°C for 3 days (3D) and 7 days (7D, or 1 week (1W)). Tris buffer showed the best performance under freeze-thaw conditions in maintaining LNP physical stability compared to other buffers, as shown by the smallest change to LNP diameter (FIG. 7A) and percent of encapsulation (FIG. 7B). Under thermal conditions (40°C), LNP physical properties such as LNP size (FIG. 7A) and percent of encapsulation (FIG. 7B) had minimal change. mRNA in LNP were chemically unstable and showed different degradation rate in different buffers, as shown by the decrease of mRNA integrity derived from the percent of main peak (FIG. 7C). Relative mRNA integrity calculated from the percentage of main peak relative to time zero (TO) was also shown (FIG. 7D). SP: Sodium Phosphate. Among all buffer / pH conditions, 2A, 2C, 4A, 4B, 4C, 5A, and 5B have the slowest mRNA degradation rate. Taken together, the use of tris pH7.5 was best in supporting LNP stability under both F / T and thermal stability at 40 °C.

[0142] Table 7 indicates calculated reaction rate constants for mRNA inside LNP when assembled under different conditions. Reaction rates are calculated based on first order kinetics.Tris (pH 7.5) is shown to be the condition with the most mRNA stability.

[0143] Table 7:Attorney Reference: RDWD-054WO

[0144] Example 6: Impact of stabilizing agents on mRNA and lipid nanoparticle freeze-thaw stability

[0145] Another set of lipid nanoparticle formulations were prepared and tested for LNP stability. Table 8 below enlists the different lipid nanoparticle formulations.

[0146] Table 8:Attorney Reference: RDWD-054WO

[0147] FIG. 8 illustrates additional stabilizing excipients in maintaining LNP stability during freeze / thaw conditions. LNP samples using nucleic acid polyA as the cargo with different cryoprotectants were frozen at -80°C overnight followed by thawing at 2-8°C for 4-6 hours. Samples were analyzed following one (1XFT) and three (3XFT) freeze-thaw cycles. LNP samples with % encapsulation higher than 80% after 3XFT underwent two additional F / T cycles for a total of five (5XFT) freeze-thaw cycles. Stabilizing agent glycerol and stabilizing agent DMSO showed best performance during freeze-thaw conditions in maintaining LNP stability compared to other excipients, as shown by the smallest change to LNP diameter (FIG. 8A) and percent of encapsulation (FIG. 8B). Glycerol and DMSO supported the freeze-thaw of LNPs beyond 5 cycles.

[0148] Example 7: Impact of the different ionizable lipids, N / P ratios and stabilizing excipients on LNP freeze-thaw stability

[0149] Another set of lipid nanoparticle formulations were prepared and tested for LNP stability. Table 9 below enlists the different lipid nanoparticle formulations.Attorney Reference: RDWD-054WO

[0151] FIG. 9 demonstrates the impact of the different ionizable lipids, N / P ratios and stabilizing excipients on LNP freeze-thaw stability. LNP samples were frozen at -80°C or -20°C overnight followed by thawing at 2-8°C for 4-6 hours. Samples were analyzed following oneAttorney Reference: RDWD-054WO(1XFT) and three (3XFT) freeze-thaw cycles. MC3 favored -20C freezing especially with sucrose or no excipient, ALC-315 favored -80C freezing especially with sucrose or no excipient; N:P ratio 3 for MC3 system produced LNPs with lower encapsulation; the dependence of different LNPs on freezing process when using sucrose was not predictable and desirable. Glycerol and DMSO formulations reduced the freezing process dependence from LNPs, and were more desirable for LNPs when freeze-thaw is necessary.

[0152] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0153] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) is not invoked.

Claims

Attorney Reference: RDWD-054WOCLAIMSWhat Is Claimed Is:

1. A lipid nanoparticle formulation, comprising: one or more nucleic acids; a lipid nanoparticle encapsulating one or more nucleic acids; wherein the lipid nanoparticle comprises lipid components, a first buffer solution having a pH in the range of 4 to 6.5, and a stabilizing agent; wherein the stabilizing agent comprises glycerol and the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

2. The formulation of claim 1, further comprising a second buffer solution having a pH in the range of 6 to 8.

3. The formulation of claim 2, wherein the second buffer solution dilutes the first buffer solution.

4. The formulation of claim 1, wherein the first buffer solution comprises sodium citrate or citric acid.

5. The formulation of claim 1, wherein the second buffer solution comprises tris(hydroxymethyl)aminomethane or its salt form.

6. The formulation of claim 1, wherein the nucleic acid is selected from a group consisting of mRNA, saRNA (self-amplifying), circular RNA, DNA, siRNA, and miRNA.

7. The formulation of claim 1, wherein the nucleic acid is in the range from about 0.01 mg / mL to about 2 mg / mL.

8. The formulation of claim 1, wherein the lipid components are selected from a group consisting of an ionizable cationic lipid, phospholipid, a PEGylated lipid and a sterol-based lipid.

9. The formulation of claim 8, wherein the ionizable cationic lipid is selected from a group consisting of DODAP, DODMA, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA and SM-102.

10. The formulation of claim 8, wherein the phospholipid is selected from a group consisting of 1,2-DOPE, 1,2-DSPE, 1,2-DOPC, 1,2-POPC, 1,2-SOPC, and DSPC.

11. The formulation of claim 8, wherein the PEGylated lipid is selected from a group consisting of DSPE-PEG(2000), DSG-PEG (2000), DPG-PEG (2000), ALC-0159 and DMG-PEG(2000).

12. The formulation of claim 8, wherein the sterol-based lipid is selected from a group consisting of cholesterol, cholesterol sulfate, campesterol, beta-sitosterol, and stigamasterol.

13. The formulation of any one of claims 1-12, wherein the lipid nanoparticle comprisesAttorney Reference: RDWD-054WO about 30-60 mol % an ionizable cationic lipid; about 0-30 mol % a phospholipid; about 15-50 mol % a sterol-based lipid; and about 0.01-10 mol % a PEGylated lipid.

14. The formulation of claim 1, wherein the first buffer solution is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, or greater than about 25 mM.

15. The formulation of claim 2, wherein the second buffer solution is greater than about 5 mM, greater than about 10 mM, greater than about 15 mM, greater than about 20 mM, or greater than about 25 mM.

16. The formulation of claim 1, wherein upon storage for a period of at least one week at a temperature of about 40° C, the integrity of the nucleic acid changes by less than about 50%.

17. The formulation of claim 1, wherein upon storage for a period of at least four weeks at a temperature of about 25° C, the integrity of the nucleic acid changes by less than about 50%.

18. The formulation of claim 1, wherein upon storage for a period of at least four months at a temperature of about 5° C, the integrity of the nucleic acid changes by less than about 50%.

19. The formulation of claim 1, wherein following at least three rounds of freezing at a temperature of about -80° C and thawing, the formulation exhibits degradation less than about 20%.

20. The formulation of claim 1, wherein following at least three rounds of freezing at a temperature of about -20° C and thawing, the formulation exhibits degradation less than about 20%.

21. Use of the formulation of any one of claims 1-20, in the manufacture of a medicament for delivering the nucleic acid in vivo, wherein the medicament is administered intravenously, subcutaneously, intramuscularly, or by pulmonary delivery.

22. The use of claim 21, wherein the nucleic acid is selected from a group consisting of mRNA, saRNA (self-amplifying), circular RNA, DNA, siRNA, and miRNA.

23. A pharmaceutical composition comprising the lipid nanoparticle formulation of any one of claims 1-20 and a pharmaceutically acceptable carrier.

24. A method of making the lipid nanoparticle formulation of claim 1, comprising: a) mixing an aqueous solution comprising one or more nucleic acids and a first buffer solution having a pH in the range of 4 to 6.5 with a solution comprising lipid components to formAttorney Reference: RDWD-054WO a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; b) diluting lipid nanoparticle solution of step a) by adding a second buffer solution; c) exchanging the first buffer solution with the second buffer solution; d) compounding and sterilizing of the lipid nanoparticle solution to form the lipid nanoparticle formulation; e) adding a stabilizing agent in any one or more of the steps a) to d); wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

25. The method of claim 24, comprising adding the second buffer solution in step d).

26. The method of claim 24, further comprising freezing the lipid nanoparticle formulation.

27. The method of claim 24, further comprising storing the lipid nanoparticle formulation.

28. The method of claim 24, wherein the mixing step is performed with a T-junction, confined impinging jets, microfluidic mixer, vortex mixer, co-axial mixer, or crossflow mixer.

29. The method of claim 24, wherein the lipid components are selected from a group consisting of an ionizable cationic lipid, a phospholipid, a PEGylated lipid and a sterol-based lipid.

30. A method of treating or preventing a disease or disorder, comprising administering to a subject in need thereof the lipid nanoparticle formulation of any one of claims 1-20.

31. A lipid nanoparticle formulation prepared by a method comprising: mixing an aqueous solution comprising one or more nucleic acids, a first buffer solution having a pH in the range of 4 to 6.5 and a stabilizing agent with a solution comprising lipid components to form a lipid nanoparticle solution comprising one or more nucleic acids encapsulated by the lipid nanoparticle; wherein the stabilizing agent comprises glycerol, wherein the stabilizing agent is in the range of 0.1 to 30 weight percentage of the total formulation.

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