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

Abstract

The present disclosure generally relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid. The present disclosure further generally relates to a method of treating or preventing a disease, the method comprising administering to a subject in need of treatment or prevention a lipid nanoparticle composition disclosed herein.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 400,545, filed August 24, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to lipid nanoparticle (LNP) compositions or formulations for nucleic acid drugs. [Background technology]

[0003] Small molecule and protein-based drugs or pharmaceuticals have been used for several decades. Nucleic acid-based therapies have recently emerged as a promising class of drugs. These drugs contain segments of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Delivering nucleic acids to target cells has been a challenge due to the lack of suitable nucleic acid delivery vectors. Viral vectors have been used for this purpose, but the innate and adaptive immune responses against these vectors and their transgene products present substantial obstacles to their more widespread use (Shirley, Jamie L. et al. Molecular Therapy (2020) 28:709-722).

[0004] In recent years, lipid nanoparticles (LNPs) have attracted much attention as delivery vehicles for nucleic acid drugs. Patisiran is the first FDA-approved small interfering RNA (siRNA) drug encapsulated in lipid nanoparticles (Kulkarni, Jayesh A. et al. Nature Nanotechnology (2021) 16:630-643). Lipid nanoparticles offer great potential for delivering nucleic acid-based drugs to target cells. At least two messenger RNA (mRNA)-based vaccines for COVID-19 disease that use lipid nanoparticles as delivery vehicles have been commercialized, and several are in different stages of clinical trials (Vu, Mai N. et al. EBioMedine (2021) 74:103-699).

[0005] The mRNA vaccine commercialized by Moderna (SPIKEVAX®) is shipped and delivered at -20°C, and Pfizer's mRNA vaccine (COMIRNATY®) is shipped and delivered at -70°C (Fahrni, ML, et al. Journal of Pharmaceutical Policy and Practice (2022) 15:16).

[0006] These lipid nanoparticle-based nucleic acid medicines need to be stored at freezing temperatures, posing storage, transportation, and last-mile delivery challenges in many countries, especially developing countries, due to the unavailability of cold chain facilities, so successful mass administration of such products across various regions may not be feasible in the event of a health crisis.

[0007] WO2022101469 describes a lyophilized formulation containing sucrose and / or trehalose as an additional component to the lipid nanoparticle formulation to improve stability. However, the lyophilized formulation requires an additional step of reconstitution before administration and may be susceptible to medication errors (Lee, Young Hwa et al. Vaccines (Basel) (2021) 9 (2): 117).

[0008] Therefore, there is a need for new lipid nanoparticle compositions for nucleic acid-based medicines. Summary of the Invention

[0009] Accordingly, the present disclosure relates to lipid nanoparticle compositions comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0010] The present disclosure generally relates to lipid nanoparticle compositions 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. The RNA can be messenger RNA (mRNA), non-coding RNA (ncRNA), or a combination thereof. The 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.

[0011] In some embodiments, the ionizable polymer is present in an amount of 1 mole percent to 25 mole 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 thereof, or a combination thereof. The chitosan, chitosan derivative, or combination thereof may be present in an amount of 1 mole percent to 25 mole percent. In some embodiments, the cellulose derivative is present in an amount of 1 mole percent to 25 mole percent. In some embodiments, the chitosan or its derivative, or combination thereof, is present in an amount of 1 mole percent to 25 mole percent, 1 mole percent to 20 mole percent, or 1 mole percent to 15 mole percent.

[0012] In some embodiments, the cationic lipid is present in an amount of 25 to 50 mole percent. The cationic lipid may be 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-C), or 3-(N-(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-C). hol), N-(l-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (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-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (Clin-DMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-oc-tadecadienoxy)propane Cutadecadienoxy)propane (CpLin-DMA), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLin-CDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), and nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (SLP-0001).

[0013] In some embodiments, the cationic lipid comprises an ionizable lipid, which may be present in an amount of 25 mole percent to 50 mole percent.

[0014] In some embodiments, the phospholipid is present in an amount of 2 mole percent to about 20 mole percent, and the phospholipid may be 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-diundecanoyl- 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-cholesterylhemisuccinoyl-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-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16).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-pa The sphingomyelin may be selected from 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.

[0015] In some embodiments, the sterol is present in an amount of 30 mole percent to about 65 mole percent. The sterol can be cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, cholesteryl decanoate, or a derivative thereof.

[0016] In some embodiments, the PEG-phospholipid is present in an amount of 0.2 mole percent to about 2.0 mole percent. The 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 with conjugated methoxyl poly(ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), or a mixture thereof.

[0017] In some embodiments, the nucleic acid encodes an antigenic polypeptide. The antigenic polypeptide can be derived from an infectious agent, such as a viral or bacterial strain.

[0018] In some embodiments, the nucleic acid controls or regulates a cellular function.

[0019] In some embodiments, the nucleic acid comprises at least one chemical modification.

[0020] In some aspects, provided herein is a nucleic acid vaccine or medicament comprising a lipid nanoparticle composition described herein.

[0021] In some aspects, provided herein are methods for treating or preventing a disease, the methods comprising administering to a subject in need thereof a lipid nanoparticle composition described herein, or a nucleic acid vaccine or medicament described herein. In some embodiments, the disease is cancer, an infectious disease, or a disease and / or disorder ameliorated by a humoral and / or cellular immune response.

[0022] In some aspects, provided herein are methods for preparing the lipid nanoparticle compositions described herein, the methods 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.

[0023] The accompanying drawings illustrate embodiments of the invention and, together with the description, serve to explain the invention. The following drawings are offered by way of example, and not by way of limitation. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows a comparative analysis of the activity of different lipid nanoparticle compositions encapsulating luciferase mRNA stored at 23±2° C. at day 1 (24 hours after formulation) and day 10 (10 days after formulation). DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Some of the terms are briefly defined herein below. The definitions should not be construed in a limiting sense.

[0026] As used herein, the singular forms "a," "an," and "the" include the plural unless the context indicates otherwise. Likewise, any singular term used herein also refers to the plural and vice versa unless the context indicates otherwise. As used herein in the claim(s), when used in conjunction with the term "comprising," the terms "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more.

[0027] The word "comprising" or any of its forms such as "comprise" or "comprises", the word "having" or any of its forms such as "have" or "has", the word "including" or any of its forms such as "include" or "includes", or the word "containing" or any of its forms such as "contains" or "contains" are open-ended and do not exclude additional, unrecited elements or method steps.

[0028] Whenever any amount or range is recited, one of ordinary skill in the art will recognize that amounts or ranges within 10 or 20 percent of the recited value would also be appropriate and reasonable and would be expected to fall within the scope of the invention.

[0029] 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. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, proteins, adjuvants, pharmaceutical biotechnology, and biopharmaceutical manufacturing described herein are those known and commonly used in the art. The methods and techniques of the present invention are generally carried out according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0030] The terms "composition" and "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 further contain a pharmaceutical carrier or excipient, such as, but not limited to, a buffer, a stabilizer, a tonicity adjuster, a surfactant, a chelating agent, a salt, an antioxidant, a diluent, and / or a preservative, or a combination thereof.

[0031] As used herein, the term "derivative" refers to a compound that can be produced in one or more steps from another compound of similar structure. Derivatives are generally formed from a similar starting compound by attaching another molecule or atom to the starting compound.

[0032] The terms "pharmaceutical," "therapeutic," "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.

[0033] The term "prevention," when used in reference to a condition such as an infection, is art-recognized and well understood in the art, and includes administration of a composition that reduces the frequency or severity of, or delays the onset of, one or more symptoms of a medical condition in a subject compared to subjects not receiving the composition. Thus, prevention of a condition such as an infection includes, for example, reducing the frequency or severity of, one or more symptoms of a medical condition in a population of treated patients compared to a control population not receiving the treatment by, for example, a statistically significant amount and / or a clinically significant amount. Similarly, prevention of an infection includes reducing the likelihood that a patient receiving the treatment will develop the infection or related symptoms compared to patients not receiving the treatment.

[0034] As used herein, the term "stable" refers to a composition that retains an acceptable degree of physical stability, chemical stability, and / or biological activity upon storage at a given temperature for a specified period of time. Pharmaceutical stability can be measured by techniques known to those skilled in the art, such as SDS PAGE, dynamic light scattering, or immunogenicity assays. A composition may be stable even if the nucleic acid contained therein does not maintain 100% of its structure and / or function and / or biological activity after storage for a specified period of time. Under certain circumstances, 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's structure and / or function and / or biological activity after storage for a specified period of time may be considered "stable." In some embodiments, a nucleic acid can be considered "stable" if it retains 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 its structure and / or function and / or biological activity after storage for a predetermined period of time.

[0035] As used herein, a "particular 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, a specified period of time also refers to a period of 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 more. In some embodiments, a specified period of time also refers to a period of 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 more.

[0036] The terms "molar ratio," "mol ratio," "molar percent," "mol percent," "mol %," or "mol %" are used interchangeably to refer to the number of moles of a component expressed as a percentage of the total molar amount of all lipid components (such as cationic lipids, phospholipids, sterols, and PEG-lipids) and ionizable polymer component(s) present in the lipid nanoparticle compositions described herein. For example, 50 mol% cationic lipid means that 50 mol% cationic lipid is present in the lipid nanoparticle composition, with the other lipid components and ionizable polymer components together comprising the remaining 50 mol%, such that the total amount of all lipid components and ionizable polymer components comprises 100 mol%.

[0037] The terms "protein" or "peptide" and "polypeptide" are used interchangeably herein to refer to a polymer of amino acids linked through peptide bonds, but not to a specific length. The terms also include fusion proteins, muteins, analogs, or modified forms.

[0038] 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, including 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.

[0039] As used herein, the term "buffer" refers to an agent that maintains the pH of a solution within a desired range.

[0040] As used herein, the term "cell" refers to a single cell or a population of cells or multiple cells.

[0041] As used herein, the term "biologically effective amount" or "therapeutically effective amount" means an amount of an agent, e.g., a medicament, drug, therapeutic agent, prophylactic agent, diagnostic agent, composition, etc., that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, prevent, diagnose, ameliorate the symptoms of, and / or delay the onset of, the infection, disease, disorder, and / or condition. The therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient.

[0042] As used herein, the term "treating" or "treatment" includes reducing, halting, or reversing the symptoms, clinical signs, or underlying pathology of a condition, stabilizing or improving the subject's condition, or making it less likely that the subject's condition will worsen to the same extent as if the subject had not received treatment. Treatment may also be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease, with the aim of reducing the risk of developing conditions associated with the disease.

[0043] The term "subject" as used herein refers to a living mammal and may be used interchangeably with the term "patient." Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. The term does not denote a particular age or sex.

[0044] As used herein, an individual "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 exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" indicates that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or condition, as known in the art. Individuals who have one or more of these risk factors are more likely to develop a particular disease, disorder, or condition than individuals who do not have one or more of these risk factors.

[0045] As used herein, the term "disease" means the interruption, cessation, or impairment of a bodily function, system, or organ. Non-limiting examples of diseases include malignant diseases, autoimmune diseases, genetic diseases, metabolic disorders, or infectious diseases.

[0046] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0047] Lipid Nanoparticle (LNP) Composition The lipid nanoparticle compositions described herein typically comprise a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0048] In some aspects, disclosed herein are lipid nanoparticle compositions comprising a nucleic acid, a biocompatible polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0049] In some aspects, disclosed herein are lipid nanoparticle compositions comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0050] In some aspects, disclosed herein are lipid nanoparticle compositions comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid, a sterol, and a PEG-lipid.

[0051] In some aspects, disclosed herein are lipid nanoparticle compositions comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, a sterol, and a PEG-lipid.

[0052] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, a sterol in an amount between 30 mole percent and 65 mole percent, and a PEG-lipid.

[0053] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 1 mole percent and 20 mole percent, a sterol in an amount between 30 mole percent and 65 mole percent, and a PEG-lipid in an amount between 0.2 mole percent and 2 mole percent.

[0054] 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 in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0055] 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 in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[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, or a combination thereof in an amount of 1 mole percent to 25 mole percent, an ionizable lipid in an amount of 25 mole percent to 50 mole percent, a phospholipid in an amount of 2 mole percent to 20 mole percent, a sterol in an amount of 30 mole percent to 65 mole percent, and a PEG-lipid in an amount of 0.2 mole percent to 2 mole percent.

[0057] In some embodiments, the present disclosure relates to a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer selected from chitosan and / or derivatives thereof, or combinations thereof, in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0058] 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 mole percent to 15 mole percent, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0059] 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.

[0060] In some embodiments, the present disclosure relates to a method of delivering a nucleic acid, the method comprising administering a lipid nanoparticle composition comprising a nucleic acid of the present disclosure, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0061] 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.

[0062] 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.

[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 in an amount between 1 mole percent and 25 mole percent, an ionizable 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 in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, 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 of treatment or prevention a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, 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 of treatment or prevention a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 molar percent and 25 molar percent, an ionizable lipid in an amount between 25 molar percent and 50 molar percent, a phospholipid in an amount between 2 molar percent and 20 molar percent, a sterol in an amount between 30 molar percent and 65 molar percent, 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 of treatment or prevention a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 molar percent and 25 molar percent, an ionizable lipid in an amount between 25 molar percent and 50 molar percent, a phospholipid in an amount between 2 molar percent and 20 molar percent, a sterol in an amount between 30 molar percent and 65 molar percent, and a PEG-lipid in an amount between 0.2 molar percent and 2 molar percent.

[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 selected from chitosan, a chitosan derivative, a cellulose derivative, poly-L-glutamic acid, or a combination thereof in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, 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 selected from chitosan, a chitosan derivative, a cellulose derivative, or a combination thereof in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[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 of treatment or prevention 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 mole percent to 25 mole percent, an ionizable lipid in an amount of 25 mole percent to 50 mole percent, a phospholipid in an amount of 2 mole percent to 20 mole percent, a sterol in an amount of 30 mole percent to 65 mole percent, and a PEG-lipid in an amount of 0.2 mole percent to 2 mole percent.

[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 and / or its derivatives, or combinations thereof, in an amount of 1 mole percent to 25 mole percent, an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0072] In some embodiments, the present 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.

[0073] In some embodiments, the present 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.

[0074] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, 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.

[0075] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, 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 comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, a sterol, and a PEG-lipid in the manufacture of a medicament for treating or preventing a disease in a subject.

[0077] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, a sterol in an amount between 30 mole percent and 65 mole percent, and a PEG-lipid in the manufacture of a medicament for treating or preventing a disease in a subject.

[0078] In some embodiments, the present disclosure relates to the use of a lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer in an amount between 1 mole percent and 25 mole percent, an ionizable lipid in an amount between 25 mole percent and 50 mole percent, a phospholipid in an amount between 2 mole percent and 20 mole percent, a sterol in an amount between 30 mole percent and 65 mole percent, and a PEG-lipid in an amount between 0.2 mole percent and 2 mole percent in the manufacture of a medicament for treating or preventing a disease in a subject.

[0079] In some embodiments, the present disclosure relates to the use of 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 in an amount of 1 mole percent to 25 mole percent, 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.

[0080] In some embodiments, the present disclosure relates to the use of 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 mole percent to 25 mole percent, 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.

[0081] In some embodiments, the present disclosure relates to the use of 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 mole percent to 25 mole percent, an ionizable lipid in an amount of 25 mole percent to 50 mole percent, a phospholipid in an amount of 2 mole percent to 20 mole percent, a sterol in an amount of 30 mole percent to 65 mole percent, and a PEG-lipid in an amount of 0.2 mole percent to 2 mole percent in the manufacture of a medicament for treating or preventing a disease in a subject.

[0082] In some embodiments, the present disclosure relates to the use of 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 mole percent to 25 mole percent, 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.

[0083] In some embodiments, the present disclosure relates to a method of delivering a nucleic acid to a cell, the method comprising delivering a lipid nanoparticle composition to the cell, the lipid nanoparticle composition comprising a nucleic acid, an ionizable polymer, a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.

[0084] 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.

[0085] nucleic acid As used herein, the term "nucleic acid" refers to a polymer comprising two or more nucleotides, e.g., deoxyribonucleotides or ribonucleotides, in either unmodified or modified form. Nucleic acids can be single- or double-stranded, linear or circular.

[0086] As used herein, the term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide. When the term nucleotide is used in reference to RNA, it refers to a ribonucleotide, and when used in reference to DNA, it refers to a deoxyribonucleotide.

[0087] 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).

[0088] The terms "deoxyribonucleic acid" or "DNA" are used interchangeably herein and refer to a polymer of deoxyribonucleotides. DNA can be single- or double-stranded, linear or circular.

[0089] In some embodiments, the nucleic acid is selected from the group consisting of 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, adipophilin, AFP, AIM-2, ART-4, BAGE, alpha-fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin D1, Ep-CAM, EphA2, EphA3, ELF-2, FGF-5, G250, gonadotropin-releasing hormone, HER-2, intestinal carbo derived from xylesterase (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, SCRN1, SOX2, SOXIO, STEAP1, survivin (BIRC5), telomerase, TGFbetaR11, TRAG-3, TRP-1, TRP-2, TERT, or WT1;Cowpox virus, vaccinia virus, pseudocowpox virus, human herpesvirus 1, human herpesvirus 2, cytomegalovirus, human adenoviruses A-F, polyomavirus, human papillomavirus, parvovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, orthoreovirus, rotavirus, Ebola virus, parainfluenza virus, influenza viruses (e.g., H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, rubella virus, pneumovirus, human respiratory syncytial virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, lymphocytic choriomeningitis virus, coronavirus (e.g., SARS-CoV-2), enterovirus, rhinovirus, poliovirus, Viral origins such as norovirus, flavivirus, dengue virus, West Nile virus, yellow fever virus, and chickenpox; anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittacosis, cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, diphtheria, Escherichia coli 0151:H7, enterohemorrhagic E. coli, enterotoxigenic E. coli, Haemophilus influenzae, Helicobacter pylori, Legionella or from protozoa, such as, for example, Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium vivax, Plasmodium ovale, or Plasmodium vivax;

[0090] Antigens can be, but are not limited to, allergens derived from cells, cell extracts, proteins, polypeptides, peptides, peptidomimetics of polysaccharides, and other molecules, such as small molecules, lipids, glycolipids, and carbohydrates from plants, animals, fungi, insects, foods, drugs, dusts, and mites. Allergens include plant pollens (e.g., ragweed / seasonal nasal allergy), weed pollen allergens, grass pollen allergens, corn, tree pollen allergens, ryegrass, spider allergens (e.g., house dust mite allergens), storage mite allergens, cedar pollen / seasonal nasal allergy; mold / fungal spore allergens, animal allergens (e.g., allergens from dogs, guinea pigs, hamsters, gerbils, rats, mice, etc.), food allergens (e.g., shellfish, nuts, citrus fruits, wheat flour, coffee), insect allergens (e.g., fleas, cockroaches), venoms: bees, hornets (yellow Examples of haptens include, but are not limited to, environmental aerobic allergens such as insects (e.g., hornets, fire ants, bees, wasps, hornets, and fire ants), bacterial allergens (e.g., streptococcal antigens, parasitic allergens, e.g., 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., acid anhydrides and isocyanates). When a hapten is used in the compositions of the present disclosure, the hapten can be coupled to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct can initiate a humoral immune response, but the hapten itself does not induce antibody production. Non-limiting examples of haptens include aniline, urushiol (a toxin found in poison ivy), hydralazine, fluorescein, biotin, digoxigenin, and dinitrophenol.

[0091] In other embodiments, the antigen is an antigen associated with a disease (eg, Alzheimer's disease) in which it is desirable to sequester the antigen in circulation, such as, for example, an amyloid protein.

[0092] In some embodiments, the nucleic acid controls or regulates a cellular function.

[0093] As used herein, the term "cellular function" refers to various cellular or biological processes such as, but not limited to, biosynthesis, cell division, cell cycle control, cellular 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.

[0094] The terms "regulate" or "modulate" or "regulation" or "modulation" are used interchangeably herein and refer to the act of controlling a cellular or biological process or the act of exerting a modifying or regulatory influence on a cellular or biological process.

[0095] messenger RNA (mRNA) Messenger RNA (mRNA) is a polymer of ribonucleotides that encodes at least a protein, polypeptide, or peptide. Typically, mRNA contains at least a coding region, a 5'UTR, a 3'UTR, a 5' cap, and a poly(A) tail. The UTR (untranslated region) is adjacent to the coding region or open reading frame (ORF). The 5'UTR and 3'UTR are the sections of mRNA before the start codon and after the stop codon, respectively. The 5'UTR has a cap (5' cap) consisting of altered nucleotides, and the mRNA also has a polyadenylation region at its 3' end with adenine nucleotides, called a poly(A) tail.

[0096] In some embodiments, mRNA can be unmodified, modified, or a combination of both. Modifications can be in the nucleobase of the nucleotide, the sugar moiety of the nucleotide, or the phosphate of the nucleotide. In some embodiments, 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.

[0097] In some embodiments, the one or more modified nucleosides is a nucleoside analog selected from 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, or 8-oxoguanosine.

[0098] In some embodiments, the one or more modified nucleosides are 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, The uridine analog is selected from 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.

[0099] In some embodiments, the one or more modified nucleosides are cytidine analogs selected from 5-methylcytidine, C5-propynyl-cytidine, C5-methylcytidine, pseudoisocytidine, 1-methyl-pseudoisocytidine, pyrrolo-pseudoisocytidine, 4-thio-pseudoisocytidine, 4-thio-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-1 deaza-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, or combinations thereof.

[0100] Methods for making modified nucleosides are known in the art (WO2020168466, US8278036, US8691966, US8748089, US8835108, US9750824, US10232055, WO2007024708, WO2012135805, WO2013052523, WO2011012316).

[0101] 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.

[0102] In some embodiments, mRNA is obtained from a natural source (ie, isolated from a cell), produced using a recombinant expression system, or chemically synthesized.

[0103] mRNA according to the present disclosure can be synthesized via 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 contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphate, and / or an RNAse inhibitor. The exact conditions may vary depending on the specific application. Methods for producing mRNA by IVT reactions are 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).

[0104] In some embodiments, in vitro transcription occurs in a single batch. In some embodiments, the IVT reaction includes capping and tailing reactions, either co-transcriptionally or separately. A cap analog is added to the in vitro transcription reaction and incorporated at the 5' end of the mRNA during the reaction. An alternative method of capping includes adding a cap post-transcriptionally through an enzymatic reaction. A poly(A) tail can be incorporated into the DNA template sequence, and thus the poly(A) tail is incorporated into the mRNA by T7 RNA polymerase during in vitro transcription. An alternative method of tailing includes adding a poly(A) tail post-transcriptionally through 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.

[0105] The mRNA produced as a result of the IVT reaction can be purified using techniques known in the art, such as centrifugation, filtration, and / or chromatography. Purification of the mRNA can be completed before the capping and tailing step is performed or after capping and tailing. The synthesized mRNA can be purified by ethanol precipitation, filtration, or chromatography. In some embodiments, the mRNA is purified using tangential flow filtration. In some embodiments, the mRNA is purified by a chromatography step. In other embodiments, the mRNA is purified by a combination of a filtration step and a chromatography step.

[0106] In some embodiments, the suitable mRNA sequence is the mRNA sequence that encodes a protein, peptide, polypeptide, or antibody.In some embodiments, the suitable mRNA sequence is codon-optimized for efficient expression in host cells or organisms.Codon optimization typically involves modifying the naturally occurring or wild-type nucleic acid sequence that encodes a peptide, polypeptide, or protein, so as to achieve the highest possible expression of the peptide, polypeptide, protein, or antibody without changing the amino acid sequence.

[0107] Any length of mRNA can be encapsulated in lipid nanoparticles of the present disclosure.The length of mRNA used in lipid nanoparticles of the present disclosure depends on the gene product or protein or protein fragment that is incorporated into lipid nanoparticles.Therefore, mRNA can be very short, about several hundred nucleotides long, or very long, about several thousand nucleotides long. In some embodiments, the mRNA 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 length. In other embodiments, the mRNA is about 0.5-30 kb, 0.5-25 kb, or 0.5-20 kb in length. In still other embodiments, the mRNA is about 1-20 kb, 1-15 kb, or 1-10 kb in length.

[0108] In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.

[0109] In some embodiments, mRNA is self-amplifying or self-replicating.As used herein, self-amplifying or self-replicating mRNA refers to the mRNA that self-replicates when delivered into cells.This mRNA typically contains a replicase, usually derived from alphavirus, which allows the original strand of mRNA that encodes the target protein to be amplified when delivered into cells (Beissert, Tim et al.Molecular Therapy 2020 28:119-128).

[0110] The mRNA present in the lipid nanoparticle composition can be present in a biologically effective or therapeutically effective amount. In some embodiments, the biologically effective amount of mRNA present in the lipid nanoparticle composition is 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 μg, 0.1 μg to 600 μg, 0.1 μg to 550 μg, 0.1 μg to 500 μg, 0.1 μg to 450 μg, 0.1 μg to 400 μg, 0.1 μg to 350 μg, 0.1 μg to 300 μg, 0.1 μg to 200 μg, or any range therebetween. In some embodiments, the biologically effective amount of 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 therebetween.

[0111] 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, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 91 μg, 92 μg, 93 μg, 94 μg, 95 μg, 96 μg, 97 μg, 98 μg, 99 μg, 100 μg, 101 μg, 102 μg, 103 μg, 104 μg, 105 μg, 106 μg, 107 μg, 108 μg, 109 μg, 110 μg, 111 μg, 112 μg, 113 μg, 114 μg, 115 μg, 116 μg, 117 μg, 118 μg, 119 μg, 120 μg, 121 μg, 5 μg, 80 μg, 85 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 220 μg, 240 μg, 250 μg, 260 μg, 280 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, or any fraction or subfraction thereof.

[0112] 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 can belong to any organism, such as a prokaryote or eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal, or a human.

[0113] Non-coding RNA (ncRNA) The terms "non-coding RNA" or "ncRNA" are used interchangeably to refer to any RNA molecule that is generally not translated, but may in some cases be translated into polypeptides or proteins, including long non-coding RNA (lncRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). The term ncRNA also includes RNAs that encode small peptides, such as lncRNA.

[0114] In some embodiments, the nucleic acid component of the lipid nanoparticle composition comprises ncRNA. The ncRNA can be naturally occurring or wild-type. In other embodiments, the ncRNA can be synthetically produced.

[0115] In some embodiments, the ncRNA is single-stranded or double-stranded.

[0116] In some embodiments, the ncRNA is a few nucleotides to several thousand nucleotides in length.

[0117] In some embodiments, the ncRNA comprises long non-coding RNA (lncRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

[0118] In some embodiments, the long non-coding RNA (lncRNA) is greater than 200 nucleotides in length.

[0119] Deoxyribonucleic acid (DNA) Any DNA molecule capable of transcribing a gene into a cell, eg, capable of expressing a transcript, can be incorporated into the lipid nanoparticle compositions described herein.

[0120] The terms "DNA sequence" or "DNA segment" or "gene" are used interchangeably and refer to a segment or sequence of DNA that can be used to produce a transcript, which can be either a messenger RNA (mRNA) or a non-coding RNA (ncRNA), such as, for example, a long non-coding RNA (lncRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), and a non-coding RNA (ncRNA), such as a PIWI-interacting RNA (piRNA), a transfer RNA (tRNA), or a ribosomal RNA (rRNA).

[0121] In some embodiments, the DNA molecule is obtained from a natural source, while in other embodiments, the DNA molecule is produced recombinantly or synthetically.

[0122] In some embodiments, the DNA molecule is modified or unmodified, linear or circular.

[0123] In some embodiments, the DNA molecule is double-stranded or single-stranded.

[0124] In some embodiments, the DNA molecule comprises a coding sequence or a non-coding sequence.

[0125] In some embodiments, the DNA molecule is a few to several thousand nucleotides in length.

[0126] ionizable polymer As used herein, the term "polymer" refers to a compound formed from multiple repeating units called monomers. Polymers are produced through a process called polymerization, in which two or more monomers are linked through chemical bonds to form a polymer. In some embodiments, polymers are branched or unbranched. In some embodiments, polymers can be homopolymers (i.e., composed of the same type of repeating unit or monomer) or heteropolymers (i.e., composed of multiple types of repeating units or monomers). The terms heteropolymer and copolymer are used interchangeably herein.

[0127] As used herein, the term "ionizable polymer" refers to 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 is cationic (positively charged) at near acidic pH (pH 1.0 to pH 6.9) and neutral (uncharged) at near physiological pH (pH 7.0 to pH 7.5).

[0128] 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 mean a polymer that is substantially free of any adverse effects when introduced into a biological system. Such polymers are capable of undergoing degradation when introduced into a biological system and are not expected to produce significant toxicity or immunological responses.

[0129] In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer, which may be selected from chitosan, chitosan derivatives, cellulose derivatives, poly-L-lysine (PLL), poly-L-glutamic acid, protamine, polyethyleneimine, derivatives thereof, or combinations thereof.

[0130] In some embodiments, the ionizable polymer is positively charged at acidic pH, ie, pH 1.0-6.9, and neutral at near physiological pH (pH 7.0-7.5).

[0131] The percentage of ionizable polymer present in the lipid nanoparticle composition can be from about 1 mol % to about 25 mol %.

[0132] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition is between about 1 mol% and about 25 mol%, between 1 mol% and about 25 mol%, between about 1 mol% and about 24 mol%, between about 1 mol% and about 23 mol%, between about 1 mol% and about 22 mol%, between about 1 mol% and about 21 mol%, between about 1 mol% and about 20 mol%, between about 1 mol% and about 19 mol%, between about 1 mol% and about 18 mol%, between about 1 mol% and about 17 mol%, between about 1 mol% and about 16 mol%, between about 1 mol% and about 15 mol%, or any range therebetween.

[0133] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition is between about 1 mol% and about 25 mol%, between about 2 mol% and about 25 mol%, between about 3 mol% and about 25 mol%, between about 4 mol% and about 25 mol%, between about 5 mol% and about 25 mol%, between about 5 mol% and about 24 mol%, between about 5 mol% and about 23 mol%, between about 5 mol% and about 22 mol%, between about 5 mol% and about 21 mol%, between about 5 mol% and about 20 mol%, between about 5 mol% and about 19 mol%, between about 5 mol% and about 18 mol%, between about 5 mol% and about 17 mol%, between about 5 mol% and about 16 mol%, between about 5 mol% and about 15 mol%, or any range therebetween.

[0134] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition is about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, 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 fraction or subfraction thereof.

[0135] In some embodiments, preferred ionizable polymers include chitosan, chitosan derivatives, cellulose derivatives, or combinations thereof.

[0136] Chitosan Chitosan is a natural polymer composed of glucosamine units. Chemically, chitosan is poly-β-(1-4)-2-amino-2-deoxy-D-glucose. Chitosan is prepared by partial deacetylation of chitin, typically by alkaline hydrolysis. Therefore, chitosan may contain acetylated units (N-acetyl-D-glucosamine) and deacetylated units (β-(1→4)-linked D-glucosamine). Chitosan molecules typically have a degree of deacetylation greater than 60% compared to chitin. The molecular weight of chitosan typically ranges between 10 kDa and 1,000 kDa. Chitosan nanoparticles have been used for drug delivery, including delivery of nucleic acids. However, chitosan nanoparticles alone are insufficient to effectively deliver nucleic acids (Ragelle, Heloise et al. Journal of Controlled Release (2013) 172:207-218).

[0137] In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer, such as chitosan, along with a lipid component and nucleic acid.

[0138] In some embodiments, the ionizable polymer is chitosan or a derivative thereof, hi some embodiments, the ionizable polymer comprises a chitosan derivative or a dialdehyde chitosan derivative, or a combination thereof.

[0139] The chitosan or chitosan derivative used in the present disclosure may have a molecular weight of about 25 kDa to about 400 kDa.

[0140] In some embodiments, the molecular weight of chitosan or its derivative is about 25 kDa to about 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 therebetween.

[0141] The proportion of chitosan or a derivative thereof, or a combination thereof, present in the lipid nanoparticle composition can be from about 1 mol % to about 25 mol %.

[0142] In some embodiments, the percentage of chitosan or a derivative thereof, or a combination 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 therebetween.

[0143] In some embodiments, the percentage of chitosan or a derivative thereof, or a combination 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 therebetween.

[0144] In some embodiments, the proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition is from about 1 mol% to about 25 mol%, preferably from about 2 mol% to 20 mol%, and most preferably from about 5 mol% to about 15 mol%.

[0145] In some embodiments, the percentage of chitosan or a derivative thereof or a combination 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 fraction or subfraction thereof.

[0146] cellulose Cellulose is a polymer composed of linear chains of d-glucose units linked via β-1,4 glycosidic bonds. It typically contains hundreds to thousands of repeating glucose units. Native cellulose is not ideal for mRNA delivery, and therefore requires modification according to the present disclosure. In some embodiments, the cellulose is modified (Jelkmann et al. Biomacrolecules (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 small molecule drug delivery (Amalin Kavitha, K. Thomas Paul, Parambath Anilkumar, Chapter 18 - Cellulose-derived materials for drug delivery applications, Editor(s): Farq Mohammad, Hamad A. Al-Lohedan, Mohammad Jawaid, In Micro and Nano Technologies, Sustainable Nanocellulose and Nanohydrogels from Natural Sources, Elsevier, 2020, Pages 367-390, ISBN 9780128167892).

[0147] In some embodiments, the lipid nanoparticle composition comprises a cellulose derivative. The cellulose derivative can be a dialdehyde cellulose derivative.

[0148] The proportion of the cellulose derivative or dialdehyde cellulose derivative, or a combination thereof, present in the lipid nanoparticle composition can be from about 1 mol % to about 25 mol %.

[0149] In some embodiments, the percentage of cellulose derivative or dialdehyde cellulose derivative or combination thereof present in the lipid nanoparticle composition is between about 1 mol% and about 25 mol%, between 1 mol% and about 25 mol%, between about 1 mol% and about 24 mol%, between about 1 mol% and about 23 mol%, between about 1 mol% and about 22 mol%, between about 1 mol% and about 21 mol%, between about 1 mol% and about 20 mol%, between about 1 mol% and about 19 mol%, between about 1 mol% and about 18 mol%, between about 1 mol% and about 17 mol%, between about 1 mol% and about 16 mol%, between about 1 mol% and about 15 mol%, or any range therebetween.

[0150] In some embodiments, the percentage of cellulose derivative or dialdehyde cellulose derivative or combination thereof present in the lipid nanoparticle composition is from about 1 mol% to about 25 mol%, from about 2 mol% to about 25 mol%, from about 3 mol% to about 25 mol%, from about 4 mol% to about 25 mol%, from about 2 mol% to about 25 mol%, from about 2 mol% to about 24 mol%, from about 2 mol% to about 23 mol%, from about 2 mol% to about 22 mol%, from about 2 mol% to about 21 mol%, from about 2 mol% to about 20 mol%, from about 2 mol% to about 19 mol%, or from about 2 mol% to about 18 mol%, from about 2 mol% to about 17 mol%, from about 2 mol% to about 16 mol%, from about 2 mol% to about 15 mol%, or any range therebetween.

[0151] In some embodiments, the proportion of cellulose derivative or dialdehyde cellulose derivative or a combination thereof present in the lipid nanoparticle composition is from about 1 mol% to about 25 mol%, preferably from about 1 mol% to 20 mol%, and most preferably from about 1 mol% to about 15 mol%.

[0152] In some embodiments, the percentage 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 fraction or subfraction thereof.

[0153] lipid components The lipid component of the lipid nanoparticle composition can include one or more lipids, such as cationic lipids, phospholipids, sterols, and PEG-lipids.

[0154] cationic lipids Cationic lipids refer to lipids that have a net positive charge at a selected pH. Cationic lipids generally consist of a hydrophilic head group that carries the charge and a hydrophobic tail group.

[0155] Examples of cationic lipids for use in lipid nanoparticle compositions include 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-(l-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyloxypropane-3 -yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (Clin-DMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9' ,12'-octadecadienoxy)propane (CpLin-DMA), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,Examples of suitable hydroxyl groups include, but are not limited to, 31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (SLP-0001), or combinations thereof.

[0156] Cationic lipids with an amine head group are preferred cationic lipids. The amine group can be at the primary, secondary, or tertiary position. The cationic lipid can contain one (monoamine) or multiple (polyamine) such amine groups.

[0157] In some embodiments, cationic lipids are positively charged at acidic pH, i.e., pH 1.0 to pH 6.9. In certain embodiments, cationic lipids are neutral at certain pHs, i.e., near physiological pH (pH 7.0 to pH 7.5). Cationic lipids that can exist in a positively charged or neutral form depending on pH are called ionizable lipids. Preferred cationic lipids are ionizable and can therefore exist in a positively charged or neutral form depending on pH. For example, ionizable lipids can be neutral near physiological pH (pH 7.0 to pH 7.5) and cationic near acidic pH (pH 1.0 to pH 6.9).

[0158] In some embodiments, the cationic lipid present in the lipid nanoparticle composition is an ionizable lipid.

[0159] Methods for making cationic lipids and / or ionizable lipids, or for imparting the functionality of cationic lipids to behave as ionizable lipids, are 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).

[0160] The percentage of cationic lipid present in the lipid nanoparticle composition is from about 25 mol % to about 50 mol %, or any range therebetween.

[0161] In some embodiments, the percentage of cationic lipid 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 therebetween.

[0162] In some embodiments, the percentage of cationic lipid 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 fraction or subfraction thereof.

[0163] phospholipids Phospholipids are lipids that contain a hydrophilic head group bearing a phosphate group and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine backbone.

[0164] Examples of phospholipids that may be used in lipid nanoparticle compositions include 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 -Diundecanoyl-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-cholesterylhemisuccinoyl-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-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16).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 Examples of suitable sphingomyelin include, but are not limited to, 1-stearoyl-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, or a combination thereof.

[0165] The percentage of phospholipids present in the lipid nanoparticle composition is from about 2 mol % to about 20 mol %, or any range therebetween.

[0166] In some embodiments, the percentage of phospholipids present in the lipid nanoparticle composition is between about 2 mol% and about 20 mol%, between about 3 mol% and about 19 mol%, between about 3 mol% and about 18 mol%, between about 3 mol% and about 17 mol%, between about 3 mol% and about 16 mol%, between about 3 mol% and about 15 mol%, between about 3 mol% and about 14 mol%, between about 3 mol% and about 13 mol%, between about 3 mol% and about 12 mol%, or any range therebetween.

[0167] In some embodiments, the percentage 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 fraction or subfraction thereof.

[0168] sterols The lipid nanoparticle compositions disclosed herein may contain sterols and / or sterol derivatives. As used herein, the term "sterol" includes, but is not limited to, cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol, or derivatives thereof. In some embodiments, the lipid nanoparticle composition contains cholesterol and / or cholesterol derivatives. Non-limiting examples of cholesterol and cholesterol derivatives include 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, cholesteryl decanoate, or mixtures thereof. Methods for producing cholesterol and cholesterol derivatives are known in the art (WO2009127060, WO2019152557).

[0169] The percentage of sterol present in the lipid nanoparticle composition can be from about 30 mol % to about 65 mol %, or any range therebetween.

[0170] In some embodiments, the percentage of sterol present in the lipid nanoparticle composition is between about 30 mol% and about 65 mol%, between about 31 mol% and about 60 mol%, between about 32 mol% and about 60 mol%, between about 33 mol% and about 60 mol%, between about 34 mol% and about 60 mol%, between about 35 mol% and about 60 mol%, or any range therebetween.

[0171] In some embodiments, the percentage 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 fraction or subfraction thereof.

[0172] PEG-lipid The terms PEG-lipid, PEGylated lipid, PEG-linked lipid, PEG-conjugated lipid, PEG-lipid conjugate, and PEG-modified lipid are used interchangeably to refer to polyethylene glycol linked to a lipid moiety. The lipid moiety may be directly linked to a PEG molecule or may be linked via a linker. In some embodiments, the PEG-lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and / or a PEG-modified cholesterol, and / or a mixture thereof. Methods for producing PEG-lipids are known to those skilled in the art; see, for example, US20030077829, US2005008689, US5885613, US7404969, WO2005026372, and WO2009086558.

[0173] In some embodiments, the PEG-lipid is selected from 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 with attached methoxyl poly(ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), or a mixture thereof.

[0174] The PEG portion of the PEG-lipid can have an average molecular weight ranging from 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, or 0.5 kDa to 2 kDa. In preferred embodiments, the PEG-lipid has an average molecular weight of about 0.5 kDa to about 2 kDa.

[0175] The percentage of PEG-lipid present in the lipid nanoparticle composition can be from about 0.2 mol % to about 2.0 mol %, or any range therebetween.

[0176] In some embodiments, the percentage of PEG-lipid present in the lipid nanoparticle composition is from about 0.2 mol% to about 2.0 mol%, from about 0.2 mol% to about 1.8 mol%, from about 0.2 mol% to about 1.5 mol%, or any range therebetween.

[0177] In some embodiments, the percentage 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 fraction or subfraction thereof.

[0178] Treatment methods Disclosed herein is a method for treating or preventing a disease. The method may comprise administering the lipid nanoparticle composition disclosed herein to a subject in need of treatment or prevention. The disease may be cancer, infectious disease, or a disease and / or disorder that is ameliorated by humoral and / or cellular immune responses.

[0179] As used herein, the terms "cancer," "cancer cell," "tumor," and "tumor cell" (used interchangeably) refer to cells exhibiting abnormal growth characterized by a significant loss of control of cell proliferation, or immortalized cells. The term "cancer" or "tumor" includes metastatic and non-metastatic cancers or tumors. Cancer may be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors.

[0180] As referred to herein, a "humoral immune response" relates to antibody production and associated accessory processes, such as activation of T-helper 2 (Th2) cells and cytokine production, isotype switching, affinity maturation, and activation of memory cells. It also refers to antibody effector functions, such as neutralizing toxins, activating classical complement, and promoting phagocytosis and elimination of pathogens. Humoral immune responses are supported by CD4+ Th2 cells; thus, activation or generation of this cell type also represents a humoral immune response, as referred to herein.

[0181] The "humoral immune response" referred to herein can also encompass the generation and / or activation of T-helper 17 (Th17) cells. 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 thought to be developmentally distinct from Th1 and Th2 cells and are hypothesized to promote humoral immune responses, for example, providing important functions in antimicrobial immunity and protecting against infection. Their production of IL-22 is thought to stimulate epithelial cells to produce antimicrobial proteins, and their production of IL-17 may be involved in the recruitment, activation, and migration of neutrophils to protect against host infection by various bacterial and fungal species.

[0182] In some embodiments, the antigen encoded by the nucleic acid in the composition of the present disclosure can be a cancer or tumor-associated protein, for example, a membrane surface-associated cancer antigen that can be recognized by an antibody.

[0183] Cancers that may be treated and / or prevented by the use or administration of the compositions of the present disclosure include, but are not limited to, carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors. In one embodiment, cancer may be caused by a pathogen such as a virus. Viruses associated with the development of cancer are known to those skilled in the art and include, but are not limited to, human papillomavirus (HPV), John Cunningham virus (JCV), human herpesvirus type 8, Epstein-Barr virus (EBV), Merkel cell polyomavirus, hepatitis C virus, and human T-cell leukemia virus type 1. The compositions of the present disclosure may be used to either treat or prevent cancer, for example, to reduce the severity of cancer or prevent cancer recurrence. Cancers that may benefit from the compositions of the present disclosure include any malignant cells that express one or more tumor-specific antigens.

[0184] In some embodiments, the antigen may be a toxin or allergen that can be neutralized by an antibody.

[0185] In some embodiments, the antigen is an antigen associated with a disease (e.g., Alzheimer's disease) in which sequestering the antigen, such as an amyloid protein, in circulation is desirable. Thus, the compositions of the present disclosure may be suitable for use in a subject in need of treatment and / or prevention of a neurodegenerative disease, where the neurodegenerative disease is associated with expression of the antigen. The subject may have a neurodegenerative disease or may be at risk for developing a neurodegenerative disease. Neurodegenerative diseases that can be treated and / or prevented by use or administration of 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 formation of B-amyloid plaques and / or the association of tau protein in the brains of Alzheimer's disease patients (see, e.g., Goedert and Spillantini, Science, 314:777-781, 2006). Herpes simplex virus type 1 has also been suggested to play a causative role in people who carry susceptible versions of the apoE gene (Itzhaki and Wozniak, J Alzheimers Dis 13:393-405, 2008).

[0186] In some embodiments, the composition may comprise 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.

[0187] In some embodiments, the antigen can be any peptide or polypeptide capable of inducing a humoral immune response specific to a particular conformation on the targeted tumor cell.

[0188] 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. Thus, the compositions described herein can be useful for treating or preventing diseases and / or disorders that are ameliorated by a humoral immune response (e.g., including the production of B cells and antibodies). The compositions can find use in any case in which it is desirable to administer an antigen to a subject to induce a humoral immune response or antibody production.

[0189] In contrast to cellular immunity, humoral immune responses are mediated by secreted antibodies produced by cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as those on the surface of foreign substances and / or pathogens (e.g., viruses, bacteria, etc.), and flag them for destruction.

[0190] Antibodies are antigen-specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells). Binding of an antigen to an antibody expressed on the surface of a B cell can stimulate the B cell to activate, undergo mitosis, and induce an antibody response that involves terminal differentiation into plasma cells specialized in the synthesis and secretion of antigen-specific antibodies.

[0191] B cells are the sole producers of antibodies during an immune response and are therefore a key component of effective humoral immunity. In addition to producing large amounts of antibodies, B cells also function as antigen-presenting cells, propagating the immune response by presenting antigens to T cells, such as T helper CD4 or cytotoxic CD8. B cells and T cells are part of the adaptive immune response, which is essential for vaccine efficacy. During an active immune response induced by either vaccination or natural infection, antigen-specific B cells are activated and clonally proliferate. During proliferation, B cells evolve to have higher affinity for epitopes. B cell proliferation can be induced indirectly by activated T-helper cells or directly by stimulation of receptors such as Toll-like receptors (TLRs).

[0192] Antigen-presenting cells, such as dendritic cells, macrophages, and B cells, can assemble at the vaccination site and interact with the antigen and adjuvant contained in the vaccine. The adjuvant stimulates and activates the cells, while the antigen provides the target blueprint. Different types of adjuvants provide different stimulatory signals to cells. For example, poly I:C (a TLR3 agonist) can activate dendritic cells but not B cells. Adjuvants such as Pam3Cys, Pam2Cys, and FSL-1 are particularly suited to activating and initiating B cell proliferation, which is expected to promote the production of antibody responses (Moyle et al., Curr Med Chem, 2008; So., J Immunol, 2012, incorporated herein by reference in their entirety).

[0193] The compositions of the present disclosure may be able to stimulate a strong antibody response and thereby protect a subject from a disease, disorder, or illness associated with an antigen capable of inducing a humoral immune response.

[0194] This includes, but is not limited to, for example, infectious diseases, cancers with membrane surface-bound cancer antigens recognized by antibodies, diseases where it is desirable to sequester antigens in the circulation such as amyloid proteins (e.g., Alzheimer's disease), neutralizing toxins with antibodies, neutralizing viruses or bacteria with antibodies, or neutralizing allergens (e.g., pollen) for the treatment of allergies.

[0195] In some embodiments, the compositions may be administered orally, nasally, rectally, or parenterally. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, intrapulmonary, intrathecal, and topical administration. In some embodiments, the compositions are administered by intramuscular, subcutaneous, or intradermal injection.

[0196] The amount of composition used in a single treatment can vary depending on factors such as the nature of the negatively charged molecule being delivered, the type of formulation, and the size of the subject. One of ordinary skill in the art can determine the effective amount of composition to use in a particular application without undue experimentation.

[0197] Those skilled in the art will be able to 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 expressed, the disease state being prevented or treated, the age, health, weight, sex, and diet of the subject, and other clinical factors.

[0198] The subject to be treated may be a vertebrate, preferably a mammal, more preferably a human.

[0199] Preparation of lipid nanoparticle compositions The lipid nanoparticles of the composition are typically prepared by mixing (not necessarily in the same order) the nucleic acid, the ionizable polymer, and the lipid components, i.e., cationic lipid, phospholipid, sterol, and PEG-lipid.

[0200] The lipid nanoparticle composition can be prepared by mixing an aqueous phase (containing the nucleic acid and ionizable polymer) with an organic phase (containing the lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids).

[0201] In some embodiments, the pH of the aqueous phase (containing the nucleic acid and ionizable polymer) is about pH 1.0 to about pH 6.9, about pH 1.5 to about 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 pH 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 pH 6.0 to about pH 6.9, or any range therebetween.

[0202] In some embodiments, the pH of the aqueous phase (containing the nucleic acid and 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 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9 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, and about pH 6.9.

[0203] In some embodiments, the pH of the aqueous phase (containing the nucleic acid and ionizable polymer) is maintained by a buffer selected from a citrate buffer or an acetate buffer.

[0204] In some embodiments, the aqueous phase (containing nucleic acid and ionizable polymer) and the organic phase (containing lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids) are mixed using a microfluidic device or jet mixer. Such devices are commercially available from suppliers, such as the NanoAssemblr device from Precision Microsystems Inc., the Micropore Advanced Crossflow (AXF) device from Micropore Technologies, and the Knauer Impingement Jet Mixing Skid. A syringe pump or pneumatic pump can be used to inject or deliver the aqueous and organic phases into the microfluidic device or jet mixer.

[0205] In some embodiments, the aqueous phase (containing nucleic acid and ionizable polymer) and the organic phase (containing lipid components, i.e., ionizable lipids, phospholipids, sterols, and PEG-lipids) are mixed using a syringe pump in a microfluidic device. The flow rate of the aqueous phase and the flow rate ratio to the organic phase can be appropriately adjusted to allow the formation of lipid nanoparticles. The lipid nanoparticles thus formed can be subjected to one or more dilution steps, one or more buffer exchange steps, and one or more filtration steps.

[0206] 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).

[0207] In some embodiments, the ionizable polymer component of the lipid nanoparticle composition is present in a proportion 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 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 therebetween.

[0208] In some embodiments, the cationic lipid component in the lipid nanoparticle composition is present in a proportion 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 therebetween.

[0209] In some embodiments, the phospholipid component in the lipid nanoparticle composition is present in a proportion 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 therebetween.

[0210] In some embodiments, the sterol component of the lipid nanoparticle composition is present in a proportion 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 therebetween.

[0211] In some embodiments, the PEG-lipid component in the lipid nanoparticle composition is present in a proportion 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 therebetween.

[0212] In some embodiments, the lipid nanoparticle composition comprises about 1 mol% to about 25 mol% ionizable polymer, about 25 mol% to about 50 mol% cationic lipid, about 2 mol% to about 20 mol% phospholipid, about 30 mol% to about 65 mol% sterol, and about 0.2 mol% to about 2.0 mol% PEG-lipid.

[0213] The ionizable polymer, cationic lipid, phospholipid, sterol, and PEG-lipid are present in the lipid nanoparticle composition in mole percentages such that the sum of their mole percentages equals 100 percent.

[0214] In some embodiments, the particle size of the lipid nanoparticle composition can have 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 therebetween.

[0215] In some embodiments, the particle size of the lipid nanoparticle composition has 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.

[0216] 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 control or regulate cellular function.The nucleic acid can belong to any organism, such as prokaryotes or eukaryotes, unicellular organisms, multicellular organisms, viruses, bacteria, mycoplasma, protozoa, animals, or humans.

[0217] In some embodiments, the lipid nanoparticle compositions may be used to treat or prevent diseases, including but not limited to: Diseases caused by viruses belonging to the families, for example, Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabndoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae or Circoviridae. Diseases caused by bacteria of the genera Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, or Yersinia. Cancer, for example, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer.

[0218] 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. Those skilled in the art can make various modifications or adaptations to the present disclosure from the description and the illustrated embodiments and examples. Such modifications or adaptations are considered to be within the scope of the present disclosure. [Example]

[0219] Luciferase expression is one of the standard methods for determining the efficiency of nucleic acid-based lipid nanoparticle compositions.Therefore, the lipid nanoparticle compositions are prepared using mRNA that expresses luciferase.Any other nucleic acid can be used instead of luciferase mRNA.

[0220] Example 1: Preparation of lipid nanoparticle compositions / formulations Cationic lipid (octanoic acid, 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-, 1-octylnonyl ester-SLP0001), phospholipid (1,2-distearoyl-sn-glycero-3-phosphorocholine-DSPC), cholesterol, PEG-lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000-DMG-PEG2000), and ionizable polymer (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 number R1012).

[0221] Different lipid nanoparticle compositions were prepared according to the molar percentages shown in Table 1. [Table 1]

[0222] The nucleic acid (luciferase mRNA) and chitosan were dissolved in the aqueous phase (citrate buffer pH 5.2-5.6), and the lipid components were dissolved in the organic phase (100% ethanol). The aqueous (continuous) and organic (dispersed) phases were mixed in a microfluidic device (Micropore AXF Mini, Micropore Technologies) using a dual syringe pump (Microlab600 Dual Syringe Pump, Hamilton Company). The total flow rate was maintained at 30 mL / min or 60 mL / min, and the aqueous-to-organic flow ratio (FRR) was maintained at 3:1, respectively. The lipid nanoparticles were diluted 1:1 with phosphate-buffered saline (PBS), pH 7.4. The lipid nanoparticles were then buffer-exchanged with PBS using a 100 kDa filter and subjected to one or more ultrafiltration steps using 0.45 and / or 0.22 micron filters.

[0223] Example 2: Translational efficacy of lipid nanoparticle compositions by luciferase assay The translational efficacy of the lipid nanoparticle composition was estimated by luciferase expression using a luciferase assay. HEK293T cells were plated in RPMI medium (supplemented with 10% FBS and 100 units of pen-strep) at 1 × 10 per well in a 12-well plate. 5 Cells were seeded at a density of 10 μl. 10 μl of luciferase mRNA-encapsulated lipid nanoparticles (luciferase LNP-mRNA) was added to each well. The plate was incubated at 37°C with 5% CO2 for 24 hours. After incubation, the cells were lysed with lysis buffer (1x PBS containing 1% NP-40) and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected. 20 μl of the supernatant was added to a well of a black flat-bottom 96-well plate, and 100 μl of substrate (Promega Luciferase Assay Kit Catalog No. E4030) was added. Immediately after addition, the luminescence of the substrate was measured using a microplate reader (Infinite200pro, Tecan). The results are shown in Figure 1, demonstrating a significant reduction in the translational efficacy of the reference formulation compared to the experimental formulation.

[0224] Incorporation by Reference Each of the patents, published patent applications, and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0225] equivalent 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 which equivalents are intended to be encompassed by the following claims.

Claims

1. a. nucleic acid b. an ionizable polymer; c. cationic lipids; d. phospholipids, e. sterols, and f. PEG-lipid, A lipid nanoparticle composition comprising:

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), and a PIWI-interacting RNA (piRNA), a transfer RNA (tRNA), or a ribosomal RNA (rRNA), or a combination thereof.

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

6. The lipid nanoparticle composition of any one of the preceding 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 to 6, wherein the ionizable polymer is chitosan, a cellulose derivative, poly-L-lysine, poly-L-glutamic acid, and / or a derivative thereof 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. 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 mole percent to 25 mole percent.

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

11. 10. 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 mole percent to 25 mole percent, 1 mole percent to 20 mole percent, or 1 mole percent to 15 mole percent.

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

13. 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-(l-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (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 hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (Clin-DMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-oc-octadecadienoxy)propane ctadecadienoxy)propane (CpLin-DMA), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,The lipid nanoparticle composition according to any one of the preceding claims, which is 31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), and nonyl 8-[(8-heptadecan-9-yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (SLP-0001).

14. 10. The lipid nanoparticle composition of claim 1, 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 mole percent to 50 mole percent.

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

17. The phospholipid may be 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-diundecanoyl -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-cholesterylhemisuccinoyl-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-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE) , 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-s The lipid nanoparticle composition according to claim 16, which is n-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. 10. The lipid nanoparticle composition of any one of the preceding claims, wherein the sterol is present in an amount of from 30 mole percent to about 65 mole percent.

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

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

21. 10. The lipid nanoparticle composition of any one 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 with attached methoxyl poly(ethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), or a mixture thereof.

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

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

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

25. 10. The lipid nanoparticle composition of claim 1, wherein the nucleic acid comprises at least one chemical modification.

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

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

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

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

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

31. 26. A method for preparing a lipid nanoparticle composition according to any one of the preceding claims, comprising mixing an aqueous phase containing the nucleic acid and the ionizable polymer with an organic phase containing the cationic lipid, the phospholipid, the cholesterol and the PEG-lipid.

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