Lipid nanoparticle (LNP) compositions or formulations for nucleic acid therapy

Abstract

The present disclosure generally relates to lipid nanoparticle compositions comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid. Furthermore, the present disclosure relates generally to methods of treating or preventing a disease comprising administering to a subject in need thereof the lipid nanoparticle compositions described herein.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 400,545, filed on August 24, 2022, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to lipid nanoparticle (LNP) compositions or formulations for nucleic acid therapy. Background Art

[0004] For decades, drugs or treatments based on small molecules and proteins have been used. In recent years, nucleic acid-based treatments are becoming a promising class of drugs. These drugs contain a segment of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Due to the lack of suitable nucleic acid delivery vectors, it has always been a challenge to deliver nucleic acids to target cells. Viral vectors have been used for this purpose, but the innate and adaptive immune responses to these vectors and their transgenic products pose a major obstacle to their wider use (Shirley, Jamie L. et al. Molecular Therapy (2020) 28: 709-722).

[0005] In recent years, lipid nanoparticles (LNPs) have received a lot of attention as delivery vehicles for nucleic acid therapy. Patisiran is the first FDA-approved short interfering RNA (siRNA) therapeutic encapsulated in lipid nanoparticles (Kulkarni, Jayesh A. et al. Nature Nanotechnology (2021) 16: 630-643). Lipid nanoparticles have great prospects in delivering nucleic acid-based therapeutics to target cells. At least two messenger RNA (mRNA)-based vaccines for COVID-19 disease have been commercialized, using lipid nanoparticles as delivery vehicles, and several of them are in different stages of clinical trials (Vu, Mai N. et al. EBioMedicine (2021) 74: 103699).

[0006] Moderna's commercialized mRNA vaccine Shipped and delivered at -20°C, while the mRNA vaccine from Pfizer Shipped and delivered at -70°C (Fahrni, ML et al. Journal of Pharmaceutical Policy and Practice (2022) 15:16).

[0007] In health emergencies, successful large-scale administration of such products in different regions may not be possible because these lipid nanoparticle-based nucleic acid therapeutics must be stored at freezing temperatures, which poses problems in storage, transportation, and last-mile delivery in many countries, especially developing countries, due to the lack of available cold chain facilities.

[0008] WO2022101469 describes a lyophilized formulation comprising sucrose and / or trehalose as an additional component of a lipid nanoparticle formulation to improve stability. However, lyophilized formulations require an additional reconstitution step before administration and may be prone to medication errors (Lee, Young Hwa et al. Vaccines (Basel) (2021) 9 (2): 117).

[0009] Therefore, new lipid nanoparticle compositions are needed for nucleic acid-based therapeutics. Summary of the invention

[0010] Thus, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0011] The present disclosure generally relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid. In some embodiments, the nucleic acid is DNA, RNA, or a combination thereof. RNA can be messenger RNA (mRNA), non-coding RNA (ncRNA), or a combination thereof. Non-coding RNA (ncRNA) can be long non-coding RNA (lncRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), PIWI interacting RNA (piRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA), or a combination thereof.

[0012] In some embodiments, the ionizable polymer is present in an amount of 1 mol percent to 25 mol percent. The ionizable polymer may include a biocompatible polymer. The ionizable polymer may be chitosan, a cellulose derivative, poly-L-lysine, poly-L-glutamic acid, and / or a derivative or combination thereof. Chitosan, a chitosan derivative or a combination thereof may be present in an amount of 1 mol percent to 25 mol percent. In some embodiments, a cellulose derivative is present in an amount of 1 mol percent to 25 mol percent. In some embodiments, chitosan or its derivative or a combination thereof may be present in an amount of 1 mol percent to 25 mol percent, 1 mol percent to 20 mol percent, or 1 mol percent to 15 mol percent.

[0013] In some embodiments, the cationic lipid is present in an amount of 25 mol% to 50 mol%. The cationic lipid can be selected from N, N-dioleyl-N, N dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N, N, N trimethylammonium chloride (DOTMA); N, N-distearyl-N, N-dimethylammonium bromide (DDAB); N-(2,3 dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP); 3-(N-(N', N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N, N-dimethyltrifluoroethyl ammonium sulfate (DOSPA), dioctadecylamido glycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane ) propane (Clin-DMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',12'-octadecadienyloxy)propane (CpLin-DMA), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethylaminopropane The present invention relates to the preparation of the following compounds: 1,2-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptathriacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102), 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl ester (ALC-0315), and 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester (SLP-0001).

[0014] In some embodiments, the cationic lipid comprises an ionizable lipid. The ionizable lipid may be present in an amount of 25 mol percent to 50 mol percent.

[0015] In some embodiments, the phospholipid is present in an amount of 2 mol% to about 20 mol%. The phospholipid can be selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleo ...DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn Undecyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1- Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin and combinations thereof.

[0016] In some embodiments, sterol is present in an amount of 30 mol percent to about 65 mol percent. Sterol can be cholesterol, sitosterol, coprosterol, ergosterol, campesterol, stigmasterol, 5α-cholestanol, 5β-coprostanol, cholesterol-(2'-hydroxy)-ethyl ether, cholesterol-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestan, cholesterenone, 5α-cholestanone, 5β-cholestanone, cholesteryl decanoate or a derivative thereof.

[0017] In some embodiments, PEG-lipid is present in an amount of 0.2 mol percent to about 2.0 mol percent. PEG-lipid can be mPEG-dimyristoylglycerol (mPEG-DMG), mPEG-N, N-ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG-cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and conjugated methoxy poly (ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) or a mixture thereof.

[0018] In some embodiments, the nucleic acid encodes an antigenic polypeptide. The antigenic polypeptide may be derived from an infectious source, such as a viral strain or a bacterial strain.

[0019] In some embodiments, the nucleic acid regulates or modulates a cellular function.

[0020] In some embodiments, the nucleic acid comprises at least one chemical modification.

[0021] In some aspects, provided herein is a nucleic acid vaccine or therapeutic agent comprising the lipid nanoparticle composition described herein.

[0022] In some aspects, the present invention provides a method for treating or preventing a disease, the method comprising administering a lipid nanoparticle composition described herein or a nucleic acid vaccine or therapeutic agent described herein to a subject in need thereof. In some embodiments, the disease is cancer, an infectious disease, or a disease and / or condition that is improved by a humoral and / or cellular immune response.

[0023] In some aspects, provided herein is a method of preparing a lipid nanoparticle composition described herein, the method comprising mixing an aqueous phase comprising a nucleic acid and an ionizable polymer with an organic phase comprising a cationic lipid, a phospholipid, a sterol, and a PEG-lipid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings illustrate embodiments of the present invention and together with the description serve to explain the present invention.These drawings are provided by way of illustration and not by way of limitation.

[0025] Figure 1 Shown is a comparative analysis of the day 1 (24 h after formulation) and day 10 (10 days after formulation) activities of different lipid nanoparticle compositions encapsulating luciferase mRNA stored at 23±2°C. DETAILED DESCRIPTION

[0026] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art.Some terms are briefly defined below; these definitions should not be construed as limiting.

[0027] As used in this specification, the singular forms "a," "an," and "the" include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also contemplates the plural, and vice versa, unless the context dictates otherwise. As used in the claims herein, when used with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0028] It must be noted that the word "comprising" or any form thereof such as "comprise" or "comprises", "having" or any form thereof such as "have" or "has", "including" or any form thereof such as "include" or "includes", or "containing" or any form thereof such as "contains" or "contains" are open-ended and do not exclude additional unrecited elements or method steps.

[0029] Wherever any amount or range is stated, one skilled in the art will recognize that amounts or ranges within 10% or 20% of the stated value are also contemplated to be appropriate and reasonable, and are included within the scope of the invention.

[0030] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Typically, the terms and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, proteins, adjuvants, pharmaceutical biotechnology, and biopharmaceutical manufacturing described herein are those well known and commonly used in the art. The methods and techniques of the present invention are typically performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed in this specification.

[0031] The terms "composition" or "formulation" are used interchangeably to refer to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, and a lipid component (such as a cationic lipid, a phospholipid, a sterol, and a PEG-lipid). The composition may additionally contain a pharmaceutically acceptable carrier or excipient, such as, but not limited to, a buffer, a stabilizer, a tonicity modifier, a surfactant, a chelating agent, a salt, an antioxidant, a diluent, and / or a preservative, or a combination thereof.

[0032] The term "derivative" as used herein means a compound that can be produced in one or more steps from another compound of similar structure. Derivatives are usually formed from similar starting compounds by attaching another molecule or atom to the starting compound.

[0033] The terms "therapeutic," "therapeutic agent," "prophylactic," "prophylactic agent," or drug are used interchangeably to refer to a compound or composition (such as the lipid nanoparticle compositions described herein) that has a biological effect or combination of biological effects that prevents, inhibits, eliminates, or arrests the progression of a disease or other abnormal biological process in a subject (e.g., an animal or human).

[0034] The term "prevention" is art-recognized and well-known in the art when used relative to an illness such as infection, and includes administering a composition that reduces the frequency or severity of one or more symptoms of a medical illness in a subject, or delays its onset, relative to a subject that does not receive the composition. Thus, preventing an illness such as infection includes, for example, reducing the frequency or severity of one or more symptoms of a medical illness in a treated patient population relative to an untreated control population, for example, reducing a statistically and / or clinically significant amount. Similarly, preventing infection includes reducing the likelihood that a treated patient will develop an infection or related symptoms relative to an untreated patient.

[0035] As used herein, the term "stable" means that the composition retains an acceptable degree of physical stability, chemical stability and / or biological activity after being stored for a specified period of time at a given temperature. The stability of the treatment can be measured by techniques known to those skilled in the art, such as by SDS PAGE, dynamic light scattering or immunogenicity assays. Even if the nucleic acid contained therein cannot maintain its structure and / or function and / or biological activity 100% after storing a defined amount of time, the composition may still be stable. In some cases, after storing a defined amount of time, maintaining at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the nucleic acid structure and / or function and / or biological activity can be considered "stable". In some embodiments, after storing a defined amount of time, maintaining about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70% or about 70% to about 80% of the nucleic acid structure and / or function and / or biological activity can be considered "stable".

[0036] As used herein, a "specified period of time" means at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, or more. In some embodiments, the specified period of time also means at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 22 months, at least about 24 months or longer. In some embodiments, the specified period of time also means at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days or longer.

[0037] The terms "molar ratio", "mol ratio", "molar percentage", "mol percent", "mol %" or "mol %" are used interchangeably to refer to the number of moles of a component expressed as a percentage relative to the total number of moles of all lipid components (such as cationic lipids, phospholipids, sterols and PEG-lipids) and ionizable polymer components present in the lipid nanoparticle compositions described herein. For example, 50 mol% of cationic lipids means that 50 mol% of the cationic lipids are present in the lipid nanoparticle composition, while the other lipid components and the ionizable polymer component together make up the remaining 50 mol%, such that the total amount of all lipid components and ionizable polymer components makes up 100 mol%.

[0038] The terms "protein" or "peptide" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acids linked by peptide bonds, but do not imply any particular length. The terms also include fusion proteins, mutant proteins, analogs or modified forms.

[0039] The terms "antibody" and "antibodies" are used interchangeably herein and refer to any antibody or antibody fragment (whether naturally produced or recombinantly produced) that retains antigen binding activity. This includes monoclonal or polyclonal antibodies, single-chain antibodies, Fab fragments of monoclonal or polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, or nanobodies.

[0040] The term "buffer" as used herein means those agents that maintain the pH of a solution within a desired range.

[0041] The term "cell" as used herein means a single cell or a population of cells or a plurality of cells.

[0042] As used herein, the term "biologically effective amount" or "therapeutically effective amount" means that when administered to a subject suffering from or susceptible to infection, disease, disorder and / or illness, an agent (e.g., therapeutic agent, drug, therapeutic agent, prophylactic agent, diagnostic agent, composition, etc.) is sufficient to treat, prevent, diagnose, improve the symptoms of infection, disease, disorder and / or illness, and / or delay the onset of the amount. The therapeutically effective amount herein may vary according to factors such as the patient's disease state, age, sex and weight.

[0043] As used herein, the term "treating" or "treatment" includes alleviating a disease, arresting a disease, or reversing the symptoms, clinical signs, or underlying pathology of a disease to stabilize or improve the subject's disease, or reduce the likelihood that the subject's disease will worsen (as if the subject had not received treatment). Treatment can be administered to subjects who do not show signs of disease and / or only show early signs of disease, with the goal of reducing the risk of developing pathology associated with the disease.

[0044] The term "subject" as used herein refers to a living mammal and is used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the class of mammals: humans; non-human primates, such as chimpanzees and other apes and monkey species; livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. The terms do not denote a particular age or sex.

[0045] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not have developed detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with the development of a particular disease, disorder, or condition, as known in the art. An individual who has one or more of these risk factors has a higher likelihood of developing a particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0046] As used herein, the term "disease" means an interruption, cessation or disorder of a body function, system or organ. Non-limiting examples of diseases include malignant diseases, autoimmune diseases, genetic diseases, metabolic disorders or infectious diseases.

[0047] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or administration as a separate composition, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0048] Lipid Nanoparticle (LNP) Compositions

[0049] The lipid nanoparticle compositions described herein generally comprise a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0050] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a nucleic acid, a biocompatible polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0051] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0052] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid in an amount of 25 mol percent to 50 mol percent, a phospholipid, a sterol, and a PEG-lipid.

[0053] In some aspects, disclosed herein is a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid in an amount of 25 mol percent to 50 mol percent, a phospholipid in an amount of 2 mol percent to 20 mol percent, a sterol, and a PEG-lipid.

[0054] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid.

[0055] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 to 25 mol percent, an ionizable lipid in an amount of 25 to 50 mol percent, a phospholipid in an amount of 1 to 20 mol percent, a sterol in an amount of 30 to 65 mol percent, and a PEG-lipid in an amount of 0.2 to 2 mol percent.

[0056] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, poly-L-lysine, poly-L-glutamic acid, or a combination thereof, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid in an amount ranging from 1 mol% to 25 mol%.

[0057] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, poly-L-glutamic acid, or a combination thereof, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid in an amount ranging from 1 mol% to 25 mol%.

[0058] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid in an amount of 0.2 mol% to 2 mol%.

[0059] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan and / or its derivatives or combinations thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0060] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, chitosan or a chitosan derivative or a combination thereof in an amount of 5 mol% to 15 mol%, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0061] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a lipid nanoparticle composition described herein and a pharmaceutically acceptable carrier or excipient.

[0062] In some embodiments, the present disclosure relates to a method of delivering a nucleic acid, the method comprising administering a lipid nanoparticle composition of the present disclosure comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0063] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0064] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0065] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0066] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid, a sterol, and a PEG-lipid.

[0067] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol, and a PEG-lipid.

[0068] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid.

[0069] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid in an amount of 0.2 mol% to 2 mol%.

[0070] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, poly-L-glutamic acid, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0071] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0072] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition, the lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid in an amount of 0.2 mol% to 2 mol%.

[0073] In some embodiments, the present disclosure relates to a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan and / or its derivatives or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol and a PEG-lipid.

[0074] In some embodiments, the disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid in the manufacture of a medicament for treating or preventing a disease in a subject.

[0075] In some embodiments, the disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid in the manufacture of a medicament for treating or preventing a disease in a subject.

[0076] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0077] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer in an amount of 1 mol percent to 25 mol percent, an ionizable lipid in an amount of 25 mol percent to 50 mol percent, a phospholipid, a sterol, and a PEG-lipid.

[0078] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer in an amount of 1 to 25 mol percent, an ionizable lipid in an amount of 25 to 50 mol percent, a phospholipid in an amount of 2 to 20 mol percent, a sterol, and a PEG-lipid.

[0079] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid.

[0080] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, the lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid in an amount of 0.2 mol% to 2 mol%.

[0081] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, poly-L-lysine, poly-L-glutamic acid, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0082] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0083] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, the lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid in an amount of 25 mol% to 50 mol%, a phospholipid in an amount of 2 mol% to 20 mol%, a sterol in an amount of 30 mol% to 65 mol%, and a PEG-lipid in an amount of 0.2 mol% to 2 mol%.

[0084] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer selected from chitosan and / or its derivatives or combinations thereof in an amount of 1 mol% to 25 mol%, an ionizable lipid, a phospholipid, a sterol and a PEG-lipid.

[0085] In some embodiments, the disclosure relates to a method of delivering a nucleic acid to a cell, the method comprising delivering a lipid nanoparticle composition to the cell, wherein the lipid nanoparticle composition comprises a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0086] In some embodiments, the present disclosure relates to a method of preparing a lipid nanoparticle composition, the method comprising mixing an aqueous phase comprising a nucleic acid and an ionizable polymer with an organic phase comprising a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0087] Nucleic Acids

[0088] As used herein, the term "nucleic acid" means a polymer comprising two or more nucleotides, such as deoxyribonucleotides or ribonucleotides, in unmodified or modified form. Nucleic acids can be single-stranded or double-stranded, linear or circular.

[0089] The term "nucleotide" as used herein means a ribonucleotide or a deoxyribonucleotide. If the term nucleotide is used in the context of RNA, it refers to a ribonucleotide, and if used in the context of DNA, it refers to a deoxyribonucleotide.

[0090] The terms "ribonucleic acid" or "RNA" are used interchangeably herein and refer to a polymer of ribonucleotides. RNA can be single-stranded or double-stranded, linear or circular. The term RNA also includes messenger RNA (mRNA) and non-coding RNA (ncRNA).

[0091] The terms "deoxyribonucleic acid" or "DNA" are used interchangeably herein and refer to a polymer of deoxyribonucleotides. DNA can be single-stranded or double-stranded, linear or circular.

[0092] In some embodiments, the nucleic acid encodes an antigen such as, but not limited to, derived from cholera toxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, hemagglutinin, neuraminidase, influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1, Der-p-1, Der-f-1, adipose differentiation-related protein, AFP, AIM-2, ART-4, BAGE, alpha-fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cell cycle protein DIEp-CAM, EphA2, EphA3, ELF- 2, FGF-5, G250, Gonadotropin-releasing hormone, HER-2, intestinal carboxylesterase (iCE), IL13Ralpha2, MAGE-1, MAGE-2, MAGE-3, MART-1, MART-2, M-CSF, MDM-2, MMP-2, MUC-1, NY-EOS-1, MUM-1, MUM-2, MUM-3, p53, PBF, PRAME, PSA, PSMA, RAGE-1, RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2, SOXIO, STEAP1, survivin (BIRC5), telomerase, TGFbetaR1 l, TRAG-3, TRP-1, TRP-2, TERT or WT1; those derived from viruses such as vaccinia virus, vaccinia virus, pseudovaccinia virus, human herpes virus 1, human herpes virus 2, cytomegalovirus, human adenovirus AF, polyomavirus, human papillomavirus, parvovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, orthoreovirus (Orthoreovirus) virus), rotavirus, Ebola virus, parainfluenza virus, influenza virus (e.g., H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, rubella virus, pneumonia virus, human respiratory syncytial virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, hantavirus, lymphocytic choriomeningitis virus, coronavirus (e.g., SARS-CoV-2), enterovirus, rhinovirus, poliovirus, norovirus, flavivirus, dengue virus, West Nile virus, yellow fever virus, and varicella;those derived from bacteria such as Bacillus anthracis (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, diphtheria toxin, Escherichia coli 0151:H7, enterohemorrhagic Escherichia coli, toxigenic Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Legionella, Leptospira, Listeria, meningococci, Mycoplasma pneumoniae, Mycobacterium, pertussis, pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica, or those derived from protozoa such as Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi.;

[0093] Antigens may be, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, peptidomimetics of polysaccharides and other molecules (such as small molecules, lipids, glycolipids and carbohydrates) derived from plants, animals, fungi, insects, allergens of food, drugs, dust and mites. Allergens include, but are not limited to, ambient air allergens; plant pollens (e.g., ragweed / hay fever); weed pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; ryegrass; arachnid allergens (e.g., house dust mite allergens); storage mite allergens; Japanese cedar pollen / hay fever; mold / fungal spore allergens; animal allergens (e.g., dog, guinea pig, hamster, gerbil, rat, mouse, etc. allergens); food allergens (e.g., crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (e.g., fleas, cockroaches); venoms: (Hymenoptera, yellow jackets, bees, wasps, hornets, fire ants); bacterial allergens (e.g., streptococcal antigens; parasitic allergens such as roundworm antigens); viral antigens; drug allergens; hormones (e.g., insulin); enzymes (e.g., streptokinase); and drugs or chemicals that can act as incomplete antigens or haptens (e.g., anhydrides and isocyanates). When a hapten is used in the compositions of the present disclosure, it can be linked to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct is capable of initiating a humoral immune response, while the hapten itself does not induce antibody production. Non-limiting examples of haptens are aniline, urushiol (the toxin in poison ivy), hydralazine, fluorescein, biotin, digoxin, and dinitrophenol.

[0094] In other embodiments, the antigen is one associated with a disease where sequestration of the antigen in circulation is desirable, such as, for example, amyloid (eg, Alzheimer's disease).

[0095] In some embodiments, the nucleic acid regulates or modulates a cellular function.

[0096] The term "cellular function" as used herein means various cellular or biological processes, such as, but not limited to, biosynthesis, cell division, cell cycle regulation, cell metabolism, ion transport, absorption, secretion, homeostasis, replication, transcription, translation, cell signaling, endocytosis, exocytosis, phagocytosis, apoptosis, DNA replication, DNA repair, protein synthesis, gene regulation, cell repair, cell growth, cell differentiation, cell trafficking, cell proliferation, metabolic pathways, etc.

[0097]

[00136] The terms "regulate" or "modulate" or "regulation" or "modulation" are used interchangeably herein and mean the act of controlling or exerting a changing or controlling influence on a cell or a biological process.

[0098] Messenger RNA (mRNA)

[0099] Messenger RNA (mRNA) is a polymer of ribonucleotides that encodes at least one protein, polypeptide or peptide. Typically, mRNA comprises at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly(A) tail. The UTR (untranslated region) flanks the coding region or open reading frame (ORF). The 5'UTR and 3'UTR are the parts of the mRNA before the start codon and after the stop codon, respectively. The 5'UTR has a cap (5' cap) that is composed of altered nucleotides. The mRNA also contains a polyadenylation region at its 3' end that has adenine nucleotides, known as a poly(A) tail.

[0100] In some embodiments, the mRNA can be unmodified or modified or a combination of the two. The modification can be in the nucleobase of the nucleotide, or in the sugar moiety of the nucleotide, or in the phosphate of the nucleotide. In some embodiments, the unmodified mRNA can contain naturally occurring nucleosides, such as adenosine, guanosine, cytidine and uridine. The mRNA can contain one or more modified nucleosides, such as adenosine analogs, guanosine analogs, cytidine analogs or uridine analogs.

[0101] In some embodiments, the one or more modified nucleosides are nucleoside analogs selected from 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, or 8-oxoguanosine.

[0102] In some embodiments, the one or more modified nucleosides are uridine analogs selected from the group consisting of propynyl-uridine, pseudouridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 3-methyl-uridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3, 2'-O-dimethyl-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurino-4-thio-pseudouridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydro-uridine, dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-dihydro-pseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, or 4-methoxy-2-thio-pseudouridine.

[0103] In some embodiments, one or more modified nucleosides are cytidine analogs selected from the following: 5-methylcytidine, C5-propynyl-cytidine, C5-methylcytidine, pseudoisocytidine, 1-methyl-pseudoisocytidine, pyrrolo-pseudoisocytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 1-methyl-1-1 deaza-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine or a combination thereof.

[0104] Methods for making modified nucleosides are well known in the art (WO 2020168466, US8278036; US8691966; US8748089; US8835108; US9750824; US10232055; WO2007024708; WO2012135805; WO2013052523; WO2011012316).

[0105] In some embodiments, the modified nucleoside is a pseudouridine, e.g., 1-methyl-pseudouridine, 1-propynyl-pseudouridine, 1-carboxymethyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 4-methoxy-pseudouridine, or 4-methoxy-2-thio-pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, dihydro-pseudouridine, or a combination thereof.

[0106] In some embodiments, the mRNA is obtained from a natural source (ie, isolated from a cell), produced using a recombinant expression system, or chemically synthesized.

[0107] mRNA according to the present disclosure can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed with a 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 inhibitors. The exact conditions may vary depending on the specific application. Methods for making mRNA by IVT reactions are well known in the art (see, for example, Beckert, Bertrand and Masquida, Benoit Methods in Molecular Biology (2011) 703, 29-41; Brunelle, Julie L. and Green Rachel Methods in Enzymology (2013) 530, 101-114; Kamakaka, Rohinton T. and Kraus W. Lee Current Protocols in Cell Biology (1999) 11.6.1-11.6.17; Kanwal, Fariha et al. Cellular Physiology and Biochemistry (2018) 48: 1915-1927; WO2018157153; WO2020185811; WO2022082001).

[0108] In some embodiments, in vitro transcription occurs in a single batch. In some embodiments, the IVT reaction includes capping and tailing reactions co-transcriptionally or separately. A cap analog is added to the in vitro transcription reaction and will be incorporated into the 5' end of the mRNA during the reaction. Alternative methods of capping include adding the cap after transcription by an enzymatic reaction. The poly(A) tail can be incorporated into the DNA template sequence, and thus the poly(A) tail will be incorporated into the mRNA by T7 RNA polymerase during in vitro transcription. Alternative methods of tailing include adding the poly(A) tail after transcription by an enzymatic reaction. In some embodiments, the capping and tailing reactions are performed co-transcriptionally, i.e., during the IVT reaction. In some embodiments, the capping and tailing reactions are performed separately from the IVT reaction.

[0109] The mRNA produced by the IVT reaction can be purified using techniques well known in the art, such as centrifugation, filtration, and / or chromatography techniques. The purification of the mRNA can be accomplished before the capping and tailing steps are performed or after the capping and tailing steps. The synthesized mRNA can be purified by ethanol precipitation or filtration or chromatography. In some embodiments, tangential flow filtration is used to purify the mRNA. In some embodiments, the mRNA is purified by a chromatography step. In other embodiments, the mRNA is purified by a combination of filtration and chromatography steps.

[0110] In some embodiments, suitable mRNA sequences are mRNA sequences encoding proteins, peptides, polypeptides or antibodies. In some embodiments, suitable mRNA sequences are codon optimized to be effectively expressed in host cells or organisms. Codon optimization generally includes modifying the naturally occurring or wild-type nucleic acid sequence encoding a peptide, polypeptide or protein to achieve the highest possible expression of a peptide, polypeptide, protein or antibody without changing the amino acid sequence.

[0111] Any length of mRNA can be encapsulated in lipid nanoparticles of the present disclosure. The length of the mRNA used in the lipid nanoparticles of the present disclosure depends on the gene product or protein or protein fragment to be incorporated into the lipid nanoparticle. Therefore, the mRNA can be very short, with an extension length of about several hundred nucleotides, or very long, with an extension length of about several thousand nucleotides. In some embodiments, the mRNA length is about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5.5 kb 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 22 kb, 24 kb, 26 kb, 28 kb or 30 kb. In other embodiments, the mRNA length is about 0.5 to 30 kb, 0.5 to 25 kb, 0.5 to 20 kb. In still other embodiments, the mRNA is about 1 to 20 kb, 1 to 15 kb, or 1 to 10 kb in length.

[0112] In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.

[0113] In some embodiments, mRNA is self-amplified or self-replicating. As used herein, self-amplified or self-replicating mRNA means mRNA that self-replicates after delivery to a cell. Such mRNAs generally contain replicase, which is generally derived from alphaviruses, which can amplify the original mRNA chain encoding a protein of interest after delivery to a cell (Beissert, Tim et al. Molecular Therapy 2020 28: 119-128).

[0114] The mRNA present in the lipid nanoparticle composition can be present in a biologically effective amount or a therapeutically effective amount. In some embodiments, the biologically effective amount of the mRNA present in the lipid nanoparticle composition is between 0.1 μg and 1000 μg, 0.1 μg and 950 μg, 0.1 μg and 900 μg, 0.1 μg and 850 μg, 0.1 μg and 800 μg, 0.1 μg and 750 μg, 0.1 μg and 700 μg, 0.1 μg and 650, 0.1 μg and 600, 0.1 μg and 550, 0.1 μg and 500 μg, 0.1 and 450 μg, 0.1 μg and 400 μg, 0.1 μg and 350 μg, 0.1 and 300 μg, 0.1 and 200 μg, or any range therein. In some embodiments, the biologically effective amount of the mRNA present in the lipid nanoparticle composition is about 0.1 μg to 1000 μg, 0.1 μg to 950 μg, 0.1 μg to 900 μg, 0.1 μg to 850 μg, 0.1 μg to 800 μg, 0.1 μg to 750 μg, 0.1 μg to 700 μg, 0.1 μg to 650, 0.1 μg to 600, 0.1 μg to 550, 0.1 μg to 500 μg, or any range therein.

[0115] In some embodiments, the biologically effective amount of mRNA present in the lipid nanoparticle composition is 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7, μg, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg. g, 75μg, 80μg, 85μg, 90μg, 100μg, 110μg, 120μg, 130μg, 140μg, 150μg, 160μg, 170μg, 180μg, 190μg, 200μg, 220μg, 240μg, 250μg, 260μg, 280μg, 300μg, 350μg, 400μg, 450μg, 500μg, 600μg, 700μg, 800μg, 900μg, 1000μg or any part or fraction thereof.

[0116] In some embodiments, the mRNA encodes one or more proteins, one or more antibodies, or a combination thereof. The mRNA encoding one or more proteins or one or more antibodies may belong to any organism, such as a prokaryotic or eukaryotic organism, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoa, an animal, or a human.

[0117] Non-coding RNA (ncRNA)

[0118] The term "non-coding RNA" or "ncRNA" is used interchangeably to refer to any RNA molecule that is not usually translated into a polypeptide or protein but can sometimes be translated into a polypeptide or protein, and includes long non-coding RNA (lncRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), PIWI interacting RNA (piRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). The term ncRNA also includes such RNAs encoding small peptides, such as lncRNA.

[0119] In some embodiments, the nucleic acid component of the lipid nanoparticle composition comprises RNA. The ncRNA may be naturally occurring or wild-type. In other embodiments, the ncRNA may be produced synthetically.

[0120] In some embodiments, the ncRNA is single-stranded or double-stranded.

[0121] In some embodiments, the ncRNA is a few nucleotides to several thousand nucleotides in length.

[0122] In some embodiments, the ncRNA includes long noncoding RNA (lncRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), PIWI interacting RNA (piRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

[0123] In some embodiments, the long noncoding RNA (lncRNA) is greater than 200 nucleotides in length.

[0124] deoxyribonucleic acid (DNA)

[0125] Any DNA molecule capable of transferring a gene into a cell (eg, to express a transcript) can be incorporated into the lipid nanoparticle compositions described herein.

[0126] The terms "DNA sequence" or "DNA fragment" or "gene" are used interchangeably and mean a DNA fragment or sequence that can be used to produce a transcript, which can be a messenger RNA (mRNA) or a non-coding RNA (ncRNA), such as a long non-coding RNA (lncRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), and a PIWI-interacting RNA (piRNA), a transfer RNA (tRNA), or a ribosomal RNA (rRNA).

[0127] In some embodiments, the DNA molecules are obtained from natural sources. In other embodiments, the DNA molecules are recombinantly or synthetically produced.

[0128] In some embodiments, the DNA molecules are modified or unmodified, linear or circular.

[0129] In some embodiments, the DNA molecule is double-stranded or single-stranded.

[0130] In some embodiments, the DNA molecule comprises a coding sequence or a non-coding sequence.

[0131] In some embodiments, the DNA molecule is a few nucleotides to several thousand nucleotides in length.

[0132] Ionizable polymers

[0133] As used herein, the term "polymer" means a compound formed by a plurality of repeating units called monomers. A polymer is produced by a process called polymerization, in which two or more monomers are linked by chemical bonds to form a polymer. In some embodiments, a polymer is branched or unbranched. In some embodiments, a polymer may be a homopolymer, i.e., composed of the same type of repeating units or monomers, or a heteropolymer, i.e., composed of more than one type of repeating units or monomers. The terms "heteropolymer" and "copolymer" are used interchangeably herein.

[0134] As used herein, the term "ionizable polymer" means a polymer that can exist in a positively charged or neutral form depending on the pH of the solution or environment, for example, an ionizable polymer will be cationic (positively charged) around acidic pH (pH 1.0 to pH 6.9) and will be neutral (no charge) around physiological pH (pH 7.0 to pH 7.5).

[0135] In some embodiments, the ionizable polymer is a biocompatible polymer or a biodegradable polymer. The terms "biocompatible polymer" and "biodegradable polymer" are used interchangeably to refer to polymers that have substantially no deleterious effects when introduced into a living system or biological system. Such polymers are capable of undergoing degradation when introduced into a living system or biological system and are not expected to produce significant toxic or immune responses.

[0136] In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer. The ionizable polymer can be selected from chitosan, chitosan derivatives, cellulose derivatives, poly-L-lysine (PLL), poly-L-glutamic acid, protamine, polyethyleneimine, derivatives thereof, or combinations thereof.

[0137] In some embodiments, the ionizable polymer is positively charged at acidic pH (ie, pH 1.0 to 6.9) and is neutral around physiological pH (pH 7.0 to 7.5).

[0138] The ionizable polymer may be present in the lipid nanoparticle composition in a proportion of about 1 mol % to about 25 mol %.

[0139] In some embodiments, the proportion of ionizable polymer present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, 1 mol% to about 25 mol%, about 1 mol% to about 24 mol%, about 1 mol% to about 23 mol%, about 1 mol% to about 22 mol%, about 1 mol% to about 21 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 19 mol%, or about 1 mol% to about 18 mol%, about 1 mol% to about 17 mol%, about 1 mol% to about 16 mol%, about 1 mol% to about 15 mol%, or any range therein.

[0140] In some embodiments, the proportion of ionizable polymer present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, about 2 mol% to about 25 mol%, about 3 mol% to about 25 mol%, about 4 mol% to about 25 mol%, about 5 mol% to about 25 mol%, about 5 mol% to about 24 mol%, about 5 mol% to about 23 mol%, about 5 mol% to about 22 mol%, about 5 mol% to about 21 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 19 mol%, or about 5 mol% to about 18 mol%, about 5 mol% to about 17 mol%, about 5 mol% to about 16 mol%, about 5 mol% to about 15 mol%, or any range therein.

[0141] In some embodiments, the proportion of ionizable polymer present in the lipid nanoparticle composition is about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, or any part or fraction thereof.

[0142] In some embodiments, preferred ionizable polymers include chitosan, chitosan derivatives, cellulose derivatives, or combinations thereof.

[0143] Chitosan

[0144] Chitosan is a natural polymer composed of glucosamine units. Chitosan is chemically poly-β-(1-4)-2-amino-2-deoxy-D-glucose. Chitosan is prepared by partial deacetylation of chitin, usually by alkaline hydrolysis. Thus, chitosan may contain acetylated units (N-acetyl-D-glucosamine) as well as deacetylated units (β-(1→4)-linked D-glucosamine). Typically, the degree of deacetylation of chitosan molecules is greater than 60% when compared to chitin. The molecular weight of chitosan typically varies between 10 kDa and 1000 kDa. Chitosan nanoparticles have been used for drug delivery, including the delivery of nucleic acids. However, chitosan nanoparticles alone are not sufficient for effective delivery of nucleic acids (Ragelle, Journal of Controlled Release (2013) 172:207-218).

[0145] In some embodiments, lipid nanoparticle compositions comprise an ionizable polymer, such as chitosan, along with a lipid component and a nucleic acid.

[0146] In some embodiments, the ionizable polymer is chitosan or a derivative thereof. In some embodiments, the ionizable polymer comprises a chitosan derivative or a dialdehyde chitosan derivative or a combination thereof.

[0147] The molecular weight of chitosan or chitosan derivatives employed in the present disclosure may be from about 25 kDa to about 400 kDa.

[0148] In some embodiments, the molecular weight of chitosan or its derivative is about 25 kDa to 375 kDa, about 30 kDa to about 350 kDa, about 35 kDa to about 325 kDa, about 40 kDa to about 300 kDa, about 40 kDa to about 250 kDa, about 40 kDa to about 225 kDa, about 40 kDa to about 220 kDa, about 40 kDa to about 210 kDa, or about 40 kDa to about 200 kDa, or any range therein.

[0149] The proportion of chitosan or its derivative or combination thereof present in the lipid nanoparticle composition may be about 1 mol % to about 25 mol %.

[0150] In some embodiments, the proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, 1 mol% to about 25 mol%, about 1 mol% to about 24 mol%, about 1 mol% to about 23 mol%, about 1 mol% to about 22 mol%, about 1 mol% to about 21 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 19 mol%, or about 1 mol% to about 18 mol%, about 1 mol% to about 17 mol%, about 1 mol% to about 16 mol%, about 1 mol% to about 15 mol%, or any range therein.

[0151] In some embodiments, the proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, about 2 mol% to about 25 mol%, about 3 mol% to about 25 mol%, about 4 mol% to about 25 mol%, about 5 mol% to about 25 mol%, about 2 mol% to about 24 mol%, about 2 mol% to about 23 mol%, about 2 mol% to about 22 mol%, about 2 mol% to about 21 mol%, about 2 mol% to about 20 mol%, about 2 mol% to about 19 mol%, or about 2 mol% to about 18 mol%, about 2 mol% to about 17 mol%, about 2 mol% to about 16 mol%, about 2 mol% to about 15 mol%, or any range therein.

[0152] In some embodiments, the proportion of chitosan or its derivative or combination thereof present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, preferably about 2 mol% to 20 mol%, and most preferably about 5 mol% to about 15 mol%.

[0153] In some embodiments, the proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition is about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol% or any part or fraction thereof.

[0154] Cellulose

[0155] Cellulose is a polymer consisting of a linear chain of d-glucose units connected by β-1,4 glycosidic bonds. It usually contains repeating glucose units ranging from several hundred to several thousand. Natural cellulose is not ideal for mRNA delivery, and therefore requires modification according to the present disclosure. In some embodiments, cellulose is modified (Jelkmann et al. Biomacromolecules (2018) 19: 4059-4067; Lee, Hye Ji et al. International Journal of Biosciences Biochemistry and Bioinformatics (2019) 9: 134-140). Cellulose and cellulose-derived materials have been used for drug delivery of small molecules (Amalin Kavitha, K. Thomas Paul, Parambath Anilkumar, Chapter 18 - Cellulose-derived materials for drug delivery applications, Editors: Faruq Mohammad, Hamad A. Al-Lohedan, Mohammad Jawaid, In Micro and NanoTechnologies, Sustainable Nanocellulose and Nanohydrogels from Natural Sources, Elsevier, 2020, pp. 367-390, ISBN 9780128167892).

[0156] In some embodiments, the lipid nanoparticle composition comprises a cellulose derivative. The cellulose derivative can be a dialdehyde cellulose derivative.

[0157] The cellulose derivative or the dialdehyde cellulose derivative or the combination thereof may be present in the lipid nanoparticle composition in a proportion of about 1 mol % to about 25 mol %.

[0158] In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose derivatives or combinations thereof present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, 1 mol% to about 25 mol%, about 1 mol% to about 24 mol%, about 1 mol% to about 23 mol%, about 1 mol% to about 22 mol%, about 1 mol% to about 21 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 19 mol%, or about 1 mol% to about 18 mol%, about 1 mol% to about 17 mol%, about 1 mol% to about 16 mol%, about 1 mol% to about 15 mol%, or any range therein.

[0159] In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose derivatives or combinations thereof present in the lipid nanoparticle composition is about 1 mol% to about 25 mol%, about 2 mol% to about 25 mol%, about 3 mol% to about 25 mol%, about 4 mol% to about 25 mol%, about 2 mol% to about 25 mol%, about 2 mol% to about 24 mol%, about 2 mol% to about 23 mol%, about 2 mol% to about 22 mol%, about 2 mol% to about 21 mol%, about 2 mol% to about 20 mol%, about 2 mol% to about 19 mol%, or about 2 mol% to about 18 mol%, about 2 mol% to about 17 mol%, about 2 mol% to about 16 mol%, about 2 mol% to about 15 mol%, or any range therein.

[0160] In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose derivatives or a combination thereof present in the lipid nanoparticle composition is about 1 mol % to about 25 mol %, preferably about 1 mol % to about 20 mol %, and most preferably about 1 mol % to about 15 mol %.

[0161] In some embodiments, the proportion of cellulose or its derivatives, or combinations thereof present in the lipid nanoparticle composition is about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, or any part or fraction thereof.

[0162] Lipid components

[0163] The lipid component of the lipid nanoparticle composition may comprise one or more lipids, such as cationic lipids, phospholipids, sterols, and PEG-lipids.

[0164] Cationic lipids

[0165] Cationic lipids are lipids with a net positive charge at a selected pH. Cationic lipids are generally composed of a charged hydrophilic head group and a hydrophobic tail.

[0166] Exemplary cationic lipids for use in lipid nanoparticle compositions include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol); N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamide 1,2-Dimethylaminopropane (DLin-DMA), 1,2-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1-diol), 1,2-dimethylamino-3-(1,2-dioleyloxy)propylamine (DODMA), 1,2-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1-diol), 1,2-dimethylamino-3-(1,2-dioleyloxy)propylamine (DODMA), 1,2-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1-diol), 1,2-dimethylamino-3-(1,2-dioleyloxy)propylamine (DODMA), 1,2-dimethylamino-3-(1,2-dioleyloxy)propylamine (DODMA), 1,2-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1 2-octadecadienyloxy)propane (Clin-DMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',12'-octadecadienyloxy)propane (CpLin-DMA), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethyl Aminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptathriacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102), 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl ester (ALC-0315), 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester (SLP-0001) or a combination thereof.

[0167] Cationic lipids with amine head groups are preferred cationic lipids. The amine groups may be located at primary, secondary or tertiary positions. The cationic lipids may contain one (monoamine) or multiple (polyamine) such amine groups.

[0168] In some embodiments, the cationic lipid is positively charged at acidic pH, i.e., pH 1.0 to pH 6.9. In certain embodiments, the cationic lipid is neutral at certain pH, i.e., at physiological pH (pH 7.0 to pH 7.5). Cationic lipids that can exist in positively charged or neutral form according to pH are called ionizable lipids. Preferred cationic lipids are ionizable, so that they can exist in positively charged or neutral form according to pH. For example, ionizable lipids can be neutral at physiological pH (pH 7.0 to pH 7.5), and can be cationic at acidic pH (pH 1.0 to pH 6.9).

[0169] In some embodiments, the cationic lipid present in the lipid nanoparticle composition is an ionizable lipid.

[0170] Methods for making cationic lipids and / or ionizable lipids or conferring the ability of cationic lipids to act as ionizable lipids are well known in the art (WO2005121348; WO2009127060; WO2009086558; WO2010042877; WO2010144740; WO2011075656; WO2017049245; WO2017075531; WO2018118102; WO2015199952; Reynier P. et al. Journal of Drug Targeting (2004) 12: 25-38; Sabnis, Staci et al. Molecular Therapy (2018) 26: 1509-1519).

[0171] The cationic lipid is present in the lipid nanoparticle composition in a proportion of about 25 mol % to about 50 mol % or any range therein.

[0172] In some embodiments, the proportion of cationic lipids present in the lipid nanoparticle composition is about 25 mol% to about 50 mol%, about 25 mol% to about 48 mol%, about 25 mol% to about 46 mol%, about 25 mol% to about 45 mol%, about 25 mol% to about 44 mol%, about 25 mol% to about 43 mol%, about 25 mol% to about 42 mol%, about 25 mol% to about 41 mol%, about 25 mol% to about 40 mol%, or any range therein.

[0173] In some embodiments, the proportion of cationic lipids present in the lipid nanoparticle composition is about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%, or any part or fraction thereof.

[0174] Phospholipids

[0175] Phospholipids include lipids containing a hydrophilic head with a phosphate group and a hydrophobic tail consisting of a fatty acid chain attached to a glycerol or sphingosine backbone.

[0176] The lipid nanoparticle composition according to claim 16, wherein the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DS ...DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1 PC), 1,2-diundecyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1- Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin and combinations thereof.

[0177] The phospholipids may be present in the lipid nanoparticle composition in a ratio of about 2 mol% to about 20 mol% or any range therein.

[0178] In some embodiments, the proportion of phospholipids present in the lipid nanoparticle composition is about 2 mol% to about 20 mol%, about 3 mol% to about 19 mol%, about 3 mol% to about 18 mol%, about 3 mol% to about 17 mol%, about 3 mol% to about 16 mol%, about 3 mol% to about 15 mol%, about 3 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 3 mol% to about 12 mol%, or any range therein.

[0179] In some embodiments, the proportion of phospholipids present in the lipid nanoparticle composition is about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol% or about 20 mol% or any part or fraction thereof.

[0180] Sterols

[0181] The lipid nanoparticle compositions disclosed herein may include sterols and / or sterol derivatives. The term "sterol" as used herein includes, but is not limited to, cholesterol, sitosterol, fecal sterol, ergosterol, campesterol, stigmasterol, or derivatives thereof. In some embodiments, the lipid nanoparticle compositions include cholesterol and / or cholesterol derivatives. Non-limiting examples of cholesterol and cholesterol derivatives include 5α-cholestanol, 5β-fecal stanol, cholesterol-(2'-hydroxy)-ethyl ether, cholesterol-(4'-hydroxy)-butyl ether, 6-ketocholesterol, 5α-cholestanol, cholesterenone, 5α-cholestanone, 5β-cholestanone, decanoic acid cholesterol ester, or mixtures thereof. Methods for making cholesterol and cholesterol derivatives are well known in the art (WO2009127060; WO2019152557).

[0182] The sterol may be present in the lipid nanoparticle composition in a ratio of about 30 mol % to about 65 mol % or any range therein.

[0183] In some embodiments, the proportion of sterol present in the lipid nanoparticle composition is about 30 mol% to about 65 mol%, about 31 mol% to about 60 mol%, about 32 mol% to about 60 mol%, about 33 mol% to about 60 mol%, about 34 mol% to about 60 mol%, about 35 mol% to about 60 mol%, or any range therein.

[0184] In some embodiments, the proportion of sterol present in the lipid nanoparticle composition is about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, or any part or fraction thereof.

[0185] PEG-lipid

[0186] The terms PEG-lipid, PEGylated lipid, PEG-linked lipid, PEG-conjugated lipid, PEG-lipid conjugate, PEG-modified lipid are used interchangeably to refer to the polyethylene glycol connected to the lipid portion. The lipid portion can be directly connected to the PEG molecule or connected through a linker. In some embodiments, the PEG-lipid includes PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and / or PEG-modified cholesterol and / or a mixture thereof. The method of making PEG-lipid is well known to those skilled in the art, see, for example, US20030077829; US2005008689; US5885613; US7404969; WO2005026372; WO2009086558).

[0187] In some embodiments, the PEG-lipid is selected from mPEG-dimyristoylglycerol (mPEG-DMG), mPEG-N,N-ditetradecanoylacetamide (mPEG-DTA or ALC0159), mPEG-cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), or a mixture thereof.

[0188] The PEG portion of the PEG-lipid can have an average molecular weight in the range of 0.5 kDa to 10 kDa. In some embodiments, the PEG-lipid has an average molecular weight of about 0.5 kDa to 5 kDa, about 0.5 kDa to 4 kDa, 0.5 kDa to 3 kDa, 0.5 kDa to 2 kDa. In a preferred embodiment, the PEG-lipid has an average molecular weight of about 0.5 kDa to about 2 kDa.

[0189] The proportion of PEG-lipid present in the lipid nanoparticle composition may be about 0.2 mol % to about 2.0 mol % or any range therein.

[0190] In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle composition is about 0.2 mol% to about 2.0 mol%, about 0.2 mol% to about 1.8 mol%, about 0.2 mol% to about 1.5 mol%, or any range therein.

[0191] In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle composition is about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol% or about 2.0 mol%, or any part or fraction thereof.

[0192] Treatment

[0193] Disclosed herein are methods for treating or preventing a disease. The method may include administering a lipid nanoparticle composition disclosed herein to a subject in need thereof. The disease may be cancer, an infectious disease, or a disease and / or condition that is improved by a humoral and / or cellular immune response.

[0194] As used herein, the terms "cancer," "cancer cell," "tumor," and "tumor cell" (used interchangeably) refer to cells that exhibit abnormal growth, characterized by significantly uncontrolled cell proliferation or cells that have become immortalized. The terms "cancer" or "tumor" include metastatic as well as non-metastatic cancers or tumors. Cancer, including the presence of a malignancy, can be diagnosed using criteria generally accepted in the art.

[0195] As referred to herein, "humoral immune response" relates to antibody production and its attendant accessory processes, such as, for example, T helper cell 2 (Th2) activation and cytokine production, isotype switching, affinity maturation and memory cell activation. Humoral immune response also refers to the effector functions of antibodies, such as, for example, toxin neutralization, classical complement activation and phagocytosis promotion and pathogen elimination. Humoral immune response is assisted by CD4+Th2 cells, so the activation or generation of this cell type also indicates a humoral immune response as referred to herein.

[0196] As referred to herein, "humoral immune response" may also encompass the generation and / or activation of T helper cells 17 (Th17). Th17 cells are a subset of helper effector T lymphocytes characterized by the secretion of host defense cytokines such as IL-17, IL-17F, IL-21, and IL-22. Th17 cells are considered developmentally distinct from Th1 and Th2 cells and are hypothesized to contribute to humoral immune responses, such as providing important functions in antimicrobial immunity and protection against infection. The IL-22 they produce is thought to stimulate epithelial cells to produce antimicrobial proteins, while the production of IL-17 may be involved in the recruitment, activation, and migration of neutrophils to protect the host from infection by a variety of bacteria and fungi.

[0197] In some embodiments, the antigen encoded by the nucleic acid in the compositions of the present disclosure may be a cancer or tumor-associated protein, such as, for example, a membrane surface-bound cancer antigen that can be recognized by an antibody.

[0198] Cancers that can be treated and / or prevented by using or applying the compositions of the present disclosure include, but are not limited to, malignancies, adenocarcinomas, lymphomas, leukemias, sarcomas, blastomas, myelomas, and germ cell tumors. In one embodiment, cancer may be caused by a pathogen such as a virus. Viruses associated with cancer development are known to technicians, and include, but are not limited to, human papillomavirus (HPV), John Cunningham virus (JCV), human herpes virus 8, Epstein-Barr virus (EBV), Merkel cell polyomavirus, hepatitis C virus, and human T-cell leukemia virus-1. The compositions of the present disclosure can be used to treat or prevent cancer, such as to reduce the severity of cancer or to prevent cancer recurrence. Cancers that can benefit from the compositions of the present disclosure include any malignant cells that express one or more tumor-specific antigens.

[0199] In some embodiments, the antigen may be a toxin or allergen that can be neutralized by an antibody.

[0200] In some embodiments, the antigen may be an antigen associated with a disease, where it is desirable to isolate the antigen in the circulation, such as amyloid protein (e.g., Alzheimer's disease). Therefore, the compositions of the present disclosure may be applicable to treating and / or preventing neurodegenerative diseases in subjects in need, wherein the neurodegenerative disease is associated with the expression of the antigen. The subject may suffer from a neurodegenerative disease or may be at risk of suffering from a neurodegenerative disease. Neurodegenerative diseases that can be treated and / or prevented by using or applying the compositions of the present disclosure include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). For example, Alzheimer's disease is characterized by the association of B-amyloid plaques and / or tau proteins in the brains of Alzheimer's patients (see, e.g., Goedert and Spillantini, Science, 314:777-781, 2006). Herpes simplex virus type 1 has also been suggested to play a pathogenic role in people who carry a susceptible version of the apoE gene (Itzhaki and Wozniak, J Alzheimers Dis 13:393-405, 2008).

[0201] In some embodiments, the composition may include a mixture of B cell epitopes as antigens for inducing a humoral immune response. The B cell epitopes may be linked to form a single polypeptide.

[0202] In some embodiments, the antigen can be any peptide or polypeptide capable of inducing a specific humoral immune response against a particular conformation on a targeted tumor cell.

[0203] In some embodiments, the compositions of the present disclosure can be used to induce a humoral immune response and / or a cellular immune response in a subject. Therefore, the compositions as described herein can be used to treat or prevent diseases and / or conditions that are improved by a humoral immune response (e.g., involving B cells and antibody production). The compositions can be applied to any situation where an antigen needs to be administered to a subject to induce a humoral immune response or antibody production.

[0204] In contrast to cell-mediated immunity, humoral immune responses are mediated by secretory antibodies produced in cells of the B lymphocyte lineage (B cells). Such secretory antibodies bind to antigens, such as those on the surface of foreign substances and / or pathogens (e.g., viruses, bacteria, etc.), and mark them for destruction.

[0205] Antibodies are antigen-specific glycoprotein products of a subpopulation of white blood cells called B lymphocytes (B cells). The engagement of an antigen with an antibody expressed on the surface of a B cell can induce an antibody response, which includes stimulating the B cell to activate, undergo mitosis, and finally differentiate into a plasma cell, which is specialized for the synthesis and secretion of antigen-specific antibodies.

[0206] B cells are the only producers of antibodies in the immune response process, and are therefore the key factors for effective humoral immunity. In addition to producing a large number of antibodies, B cells also serve as antigen presenting cells, and can present antigens to T cells, such as T helper CD4 or cytotoxic CD8, thereby propagating immune responses. B cells and T cells are all part of the adaptive immune response, which is essential for vaccine efficacy. In the active immune response process induced by vaccination or natural infection, antigen-specific B cells are activated and cloned. In the amplification process, B cells evolve to have higher affinity for epitopes. The proliferation of B cells can be indirectly induced by activated T helper cells, and can also be directly induced by stimulating receptors (such as Toll-like receptors (TLRs)).

[0207] Antigen presenting cells, such as dendritic cells, macrophages and B cells, are attracted to the vaccination site and can interact with the antigens and adjuvants contained in the vaccine. The adjuvant stimulates the cells to be activated, and the antigen provides a blueprint for the target. Different types of adjuvants provide different stimulation signals to the cells. For example, Poly I:C (TLR3 agonist) can activate dendritic cells, but cannot activate B cells. Adjuvants such as Pam3Cys, Pam2Cys and FSL-1 are particularly good at activating and initiating B cell proliferation, which is expected to help produce antibody responses (Moyle et al., Curr Med Chem, 2008; So., J Immunol, 2012, which are hereby incorporated by reference in their entirety).

[0208] The compositions of the present disclosure are capable of protecting a subject from a disease, disorder or condition associated with an antigen capable of inducing a humoral immune response by stimulating a strong antibody response.

[0209] This includes, but is not limited to, for example, infectious diseases, cancers involving membrane-bound cancer antigens recognized by antibodies, diseases requiring sequestration of antigens such as amyloid in the circulation (e.g., Alzheimer's disease); neutralization of toxins with antibodies; neutralization of viruses or bacteria with antibodies; or neutralization of allergens (e.g., pollen) to treat allergies.

[0210] In some embodiments, the composition can be administered orally, nasally, rectally or parenterally. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, intrapulmonary, intrathecal and topical modes of administration. In some embodiments, the composition is administered by intramuscular, subcutaneous or intradermal injection.

[0211] The amount of the composition used in a single treatment may vary depending on factors such as the nature of the negatively charged molecule to be delivered, the type of formulation, and the size of the subject. One skilled in the art will be able to determine the effective amount of the composition to be used in a particular application without undue experimentation.

[0212] A skilled artisan can determine the appropriate treatment regimen, route of administration, dosage, etc. for any particular application to achieve the desired results. Factors that may be considered include, for example, the nature of the polypeptide to be expressed; the disease state to be prevented or treated; the age, physical condition, weight, sex and diet of the subject; and other clinical factors.

[0213] The subject to be treated may be any vertebrate, preferably a mammal, more preferably a human.

[0214] Preparation of lipid nanoparticle compositions

[0215] The lipid nanoparticles of the composition are generally prepared by mixing the nucleic acid, the ionizable polymer, and the lipid components (ie, cationic lipids, phospholipids, sterols, and PEG-lipids), not necessarily in the same order.

[0216] Lipid nanoparticle compositions can be prepared by mixing an aqueous phase (comprising nucleic acids and ionizable polymers) with an organic phase (comprising lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids).

[0217] In some embodiments, the pH of the aqueous phase (comprising the nucleic acids and the ionizable polymer) is about pH 1.0 to about pH 6.9, about pH 1.5 to about pH 6.9, about pH 2.0 to about pH 6.9, about pH 2.5 to about pH 6.9, about pH 3.0 to about pH 6.9, about pH 3.5 to about pH 6.9, about pH 3.8 to about pH 6.9, about pH 4.0 to about pH 6.9, about 4.2 to about pH 6.9, about pH 4.6 to about pH 6.9, about pH 5.0 to about pH 6.9, about 6.0 to about pH 6.9, or any range therein.

[0218] In some embodiments, the pH of the aqueous phase (comprising the nucleic acids and the ionizable polymer) is about pH 2.0, about pH 2.1, about pH 2.2, about pH 2.3, about pH 2.4, about pH 2.5, about pH 2.6, about pH 2.7, about pH 2.8, about pH 2.9, about pH 3.0, about pH 3.1, about pH 3.2, about pH 3.3, about pH 3.4, about pH 3.5, about pH 3.6, about pH 3.7, about pH 3.8, about pH 3.9, about pH 4.0, about pH 4.1, about pH 4.2, about pH 4.3, about pH 4.4, about pH 4.5, about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9 about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9.

[0219] In some embodiments, the pH of the aqueous phase (comprising the nucleic acid and the ionizable polymer) is maintained by a buffer selected from a citrate buffer or an acetate buffer.

[0220] In some embodiments, a microfluidic device or jet mixer is used to mix the aqueous phase (comprising nucleic acids and ionizable polymers) and the organic phase (comprising lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids). Such devices are commercially available from suppliers, e.g., NanoAssemblr devices from Precision Microsystem Inc., Micropore Advanced Cross Flow (AXF) devices from Micropore Technologies, impact jet mixers from Knauer, etc. A syringe pump or pneumatic pressure pump can be used to inject or deliver the aqueous and organic phases to the microfluidic device or jet mixer.

[0221] In some embodiments, a syringe pump in a microfluidic device is used to mix an aqueous phase (comprising nucleic acids and ionizable polymers) and an organic phase (comprising lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids). The flow rate and flow rate ratio of the aqueous phase to the organic phase can be appropriately adjusted to enable the formation of lipid nanoparticles. The lipid nanoparticles so formed can be subjected to one or more dilution steps, one or more buffer exchange steps, and one or more filtration steps.

[0222] In some embodiments, the lipid nanoparticles formed according to the process are buffer exchanged with phosphate-buffered saline such that the final pH of the lipid nanoparticle composition is neutral (pH 7.0 to pH 7.5).

[0223] In some embodiments, the ionizable polymer component of the lipid nanoparticle composition is present in a ratio of about 1 mol% to about 25 mol%, about 1 mol% to about 24 mol%, about 1 mol% to about 23 mol%, about 1 mol% to about 22 mol%, about 1 mol% to about 21 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 19 mol%, or from about 1 mol% to about 18 mol%, about 1 mol% to about 17 mol%, about 1 mol% to about 16 mol%, about 1 mol% to about 15 mol%, or any range therein.

[0224] In some embodiments, the cationic lipid component of the lipid nanoparticle composition is present in a ratio of about 25 mol% to about 50 mol%, about 25 mol% to about 48 mol%, about 25 mol% to about 46 mol%, about 25 mol% to about 45 mol%, about 25 mol% to about 44 mol%, about 25 mol% to about 43 mol%, about 25 mol% to about 42 mol%, about 25 mol% to about 41 mol%, about 25 mol% to about 40 mol%, or any range therein.

[0225] In some embodiments, the phospholipid component of the lipid nanoparticle composition is present in a ratio of about 2 mol% to about 20 mol%, about 3 mol% to about 19 mol%, about 3 mol% to about 18 mol%, about 3 mol% to about 17 mol%, about 3 mol% to about 16 mol%, about 3 mol% to about 15 mol%, about 3 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 3 mol% to about 12 mol%, or any range therein.

[0226] In some embodiments, the sterol component of the lipid nanoparticle composition is present in a ratio of about 30 mol % to about 65 mol %, about 31 mol % to about 60 mol %, about 32 mol % to about 60 mol %, about 33 mol % to about 60 mol %, about 34 mol % to about 60 mol %, about 35 mol % to about 60 mol %, or any range therein.

[0227] In some embodiments, the PEG-lipid component of the lipid nanoparticle composition is present in a ratio of about 0.2 mol% to about 2.0 mol%, about 0.2 mol% to about 1.8 mol%, about 0.2 mol% to about 1.5 mol%, or any range therein.

[0228] In some embodiments, the lipid nanoparticle composition comprises about 1 mol % to about 25 mol % of an ionizable polymer, about 25 mol % to about 50 mol % of a cationic lipid, about 2 mol % to about 20 mol % of a phospholipid, about 30 mol % to about 65 mol % of a sterol, and about 0.2 mol % to about 2.0 mol % of a PEG-lipid.

[0229] The ionizable polymer, cationic lipid, phospholipid, sterol and PEG-lipid are present in the lipid nanoparticle composition in mol percentages such that the sum of their mol percentages is 100%.

[0230] In some embodiments, the particle size of the lipid nanoparticle composition can be an average diameter of about 10 nm to about 500 nm, about 20 nm to about 400 nm, about 30 nm to about 350 nm, about 40 nm to about 300 nm, about 50 nm to about 300 nm, about 60 nm to about 300 nm, or any range therein.

[0231] In some embodiments, the particle size of the lipid nanoparticle composition can be an average diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 260 nm, about 280 nm, about 290 nm, or about 300 nm.

[0232] The lipid nanoparticle compositions described herein can be used as a platform for therapeutic or prophylactic delivery of nucleic acids. The nucleic acid can encode one or more antigens, one or more proteins, one or more antibodies, or a combination thereof. The nucleic acid can regulate or modulate cellular function. The nucleic acid can belong to any organism, such as a prokaryotic or eukaryotic organism, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoa, an animal, or a human.

[0233] In some embodiments, lipid nanoparticle compositions can be used to treat or prevent diseases such as, but not limited to:

[0234] Diseases caused by viruses belonging to the following families: for example, Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepadnaviridae or Circoviridae.

[0235] Diseases caused by bacteria belonging to the genera Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia coli, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio or Yersinia.

[0236] Cancer, such as bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer.

[0237] The present disclosure is further illustrated by the following non-limiting examples. It should be understood that the examples are provided to illustrate the present disclosure. Based on the description and the exemplary embodiments and examples, those skilled in the art may make various modifications or adjustments to the present disclosure. Such modifications or adjustments are considered to be within the scope of the spirit of the present disclosure.

[0238] Example

[0239] The expression of luciferase is one of the standard ways to determine the efficiency of nucleic acid-based lipid nanoparticle compositions. Therefore, lipid nanoparticle compositions are prepared by expressing luciferase using mRNA. Any other nucleic acid can be used instead of luciferase mRNA.

[0240] Example 1: Preparation of lipid nanoparticle compositions / formulations

[0241] Cationic lipids (octanoic acid, 8-[(2-hydroxyethyl) [8-(nonyloxy)-8-oxooctyl] amino] -, 1-octylnonyl ester - SLP0001), phospholipids (1,2-distearoyl-sn-glycero-3-phosphocholine - DSPC), cholesterol, PEG-lipids (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 - DMG-PEG 2000), ionizable polymers (chitosan - low molecular weight) were obtained from Sapala Life Sciences Private Limited, Avanti Polar Lipids, Sigma-Aldrich, Avanti Polar Lipids and Sigma-Aldrich, respectively. Luciferase mRNA was obtained from APExBio (Catalog No. R1012).

[0242] Different lipid nanoparticle compositions were prepared according to the mol percentages given in Table 1.

[0243] Table 1

[0244]

[0245] Note: RF = reference preparation; EF = experimental preparation

[0246] Nucleic acid (luciferase mRNA) and chitosan are dissolved in aqueous phase (citrate buffer pH 5.2 to 5.6), and lipid components are dissolved in organic phase (100% ethanol). Aqueous phase (continuous phase) and organic phase (dispersed phase) are mixed using a dual syringe pump (Microlab 600 dual syringe pump, Hamilton Company) in a microfluidic device (Micropore AXF Mini, Micropore Technologies). The total flow rate is maintained at 30 mL / min or 60 mL / min, respectively, and the flow rate ratio (FRR) of the aqueous phase to the organic phase is 3: 1. Lipid nanoparticles are diluted with phosphate buffered saline (PBS) at a ratio of 1: 1 with pH 7.4. Lipid nanoparticles are then buffer exchanged with PBS using a 100 kDa filter, and ultrafiltration steps are performed using 0.45 and / or 0.22 micron filters.

[0247] Example 2: Trans-translational efficacy of lipid nanoparticle compositions as determined by luciferase

[0248] The translational efficacy of the lipid nanoparticle compositions was evaluated by luciferase expression in a luciferase assay. HEK 293T cells in RPMI medium (supplemented with 10% FBS and 100 units of penicillin-streptomycin (pen-strep)) were cultured at 1×10 5The density of cells was inoculated into 12-well plates. 10 μl of luciferase mRNA encapsulated lipid nanoparticles (luciferase LNP-mRNA) were added to each well. The plate was incubated at 37°C, 5% CO2 for 24 hours. After incubation, the cells were lysed with lysis buffer (1xPBS with 1% NP-40) and centrifuged at 8000rpm for 10 min at 4°C. The supernatant was collected. 20 μl of supernatant was added to the wells of a black flat-bottom 96-well plate, and 100 μl of substrate (Promega luciferase assay kit, catalog number E4030) was added. Luminescence was measured using a microplate reader (Infinite 200pro, Tecan) immediately after adding the substrate. The results are shown in Figure 1 , indicating that the translational efficacy of the reference formulation was substantially reduced compared to the experimental formulation.

[0249] Incorporated by Reference

[0250] Each of the patents, published patent applications, and non-patent references cited herein is hereby incorporated by reference in its entirety.

[0251] Equivalent solutions

[0252] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A lipid nanoparticle composition, comprising: a. Nucleic acids, b. ionizable polymers, c. Cationic lipids, d. phospholipids, e. sterols, and f. PEG-lipids.

2. The lipid nanoparticle composition of claim 1, wherein the nucleic acid is DNA, RNA or a combination thereof.

3. The lipid nanoparticle composition of claim 2, wherein the RNA is messenger RNA (mRNA), non-coding RNA (ncRNA), or a combination thereof.

4. The lipid nanoparticle composition of claim 3, wherein the non-coding RNA (ncRNA) is a long non-coding RNA (lncRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a PIWI-interacting RNA (piRNA), a transfer RNA (tRNA) or a ribosomal RNA (rRNA) or a combination thereof.

5. The lipid nanoparticle composition of any one of the preceding claims, wherein the ionizable polymer is present in an amount of 1 mol percent to 25 mol percent.

6. The lipid nanoparticle composition of any one of claims 1 to 5, wherein the ionizable polymer comprises a biocompatible polymer.

7. The lipid nanoparticle composition according to any one of the preceding claims 1-6, wherein the ionizable polymer is chitosan, a cellulose derivative, poly-L-lysine, poly-L-glutamic acid and / or their derivatives, or a combination thereof.

8. The lipid nanoparticle composition of claim 7, wherein the ionizable polymer is chitosan, a cellulose derivative and / or a derivative thereof, or a combination thereof.

9. The lipid nanoparticle composition of claim 8, wherein the chitosan, the chitosan derivative, or a combination thereof is present in an amount of 1 mol percent to 25 mol percent.

10. The lipid nanoparticle composition of claim 8, wherein the cellulose derivative is present in an amount of 1 mol percent to 25 mol percent.

11. The lipid nanoparticle composition of claim 9, wherein the chitosan or a derivative thereof or a combination thereof is present in an amount of 1 mol percent to 25 mol percent, 1 mol percent to 20 mol percent, or 1 mol percent to 15 mol percent.

12. The lipid nanoparticle composition of any one of the preceding claims, wherein the cationic lipid is present in an amount of 25 mol percent to 50 mol percent.

13. A lipid nanoparticle composition according to any of the preceding claims, wherein the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3 dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)-N-2- (Spermine carboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamido glycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (Clin-DMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',12'-octadecadienyloxy)propane (CpLin-DMA), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl- 4-Dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptathriacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM-102), 2-hexyldecanoic acid 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl ester (ALC-0315) and 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoic acid nonyl ester (SLP-0001).

14. The lipid nanoparticle composition of any one of the preceding claims, wherein the cationic lipid comprises an ionizable lipid.

15. The lipid nanoparticle composition of claim 14, wherein the ionizable lipid is present in an amount of 25 mol percent to 50 mol percent.

16. The lipid nanoparticle composition of any of the preceding claims, wherein the phospholipid is present in an amount of 2 mol percent to about 20 mol percent.

17. The lipid nanoparticle composition of claim 16, wherein the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine ( DSPC), 1,2-diundecyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1- Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin and combinations thereof.

18. The lipid nanoparticle composition of any one of the preceding claims, wherein the sterol is present in an amount of 30 mol percent to about 65 mol percent.

19. The lipid nanoparticle composition of any one of the preceding claims, wherein the sterol is cholesterol, sitosterol, coprosterol, ergosterol, campesterol, stigmasterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholesterenone, 5α-cholestane, 5β-cholestane, cholesteryl decanoate, or a derivative thereof.

20. The lipid nanoparticle composition of any one of the preceding claims, wherein the PEG-lipid is present in an amount of 0.2 mol percent to about 2.0 mol percent.

21. A lipid nanoparticle composition according to any of the preceding claims, wherein the PEG-lipid is mPEG-dimyristoylglycerol (mPEG-DMG), mPEG-N,N-ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG-cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxy poly(ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), or a mixture thereof.

22. The lipid nanoparticle composition of any of the preceding claims, wherein the nucleic acid encodes an antigenic polypeptide or regulates or modulates a cellular function.

23. The lipid nanoparticle composition of claim 22, wherein the antigenic polypeptide is derived from an infectious source.

24. The lipid nanoparticle composition of claim 23, wherein the infectious agent is selected from a viral strain and a bacterial strain.

25. The lipid nanoparticle composition of any of the preceding claims, wherein the nucleic acid comprises at least one chemical modification.

26. A nucleic acid vaccine comprising the lipid nanoparticle composition according to any one of the preceding claims.

27. A method for treating or preventing a disease, the method comprising administering a lipid nanoparticle composition according to any one of claims 1 to 25 or a nucleic acid vaccine according to claim 26 to a subject in need thereof.

28. The method of claim 27, wherein the disease is cancer, an infectious disease, or a disease and / or condition ameliorated by a humoral and / or cellular immune response.

29. Use of the lipid nanoparticle composition according to any one of the preceding claims 1-25 in the manufacture of a medicament for treating a disease in a subject.

30. The use according to claim 29, wherein the disease is cancer, infectious disease or a disease and / or condition that is improved by humoral and / or cellular immune response.

31. A method for preparing a lipid nanoparticle composition as described in any one of the preceding claims 1-25, the method comprising mixing an aqueous phase and an organic phase, the aqueous phase comprising the nucleic acid and the ionizable polymer, and the organic phase comprising the cationic lipid, the phospholipid, the cholesterol and the PEG-lipid.

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