Process for preparing lipid nanoparticle compositions

Abstract

Provided are empty lipid nanoparticle compositions, and processes for their preparation, that are useful for preparing therapeutic or prophylactic lipid nanoparticle compositions that contain a therapeutic or prophylactic agent, including, for example, a nucleic acid such as mRNA.

Description

[Technical field]

[0001] Provided are empty lipid nanoparticle compositions, as well as processes for their preparation and for the preparation of therapeutic or prophylactic lipid nanoparticle compositions that contain a therapeutic or prophylactic agent, including nucleic acids such as, for example, mRNA. [Background technology]

[0002] Effective targeted delivery of bioactive substances, such as small molecule drugs, proteins, and nucleic acids, is a continuing medical challenge. In particular, the delivery of nucleic acids into cells is made difficult by the relative instability and low cell permeability of such species. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and prophylactic agents, such as nucleic acids, into cells. Summary of the Invention

[0003] The present disclosure provides LNP molecules for delivering nucleic acid molecules, eg, mRNA therapeutics, for the prophylactic benefit of patients.

[0004] In certain embodiments, there is provided a process for preparing an empty lipid nanoparticle composition, comprising: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid Provided herein is a process that includes mixing with an aqueous buffered solution having a pH of less than about 4.5.

[0005] In certain embodiments, a process for preparing a loaded lipid nanoparticle composition is provided, comprising: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid with an aqueous buffer solution having a pH of less than about 4.5 to obtain a blank lipid nanoparticle composition; (b) combining the empty lipid nanoparticle composition with a payload to form a filled lipid nanoparticle composition.

[0006] In certain embodiments, provided herein are loaded lipid nanoparticles prepared by the processes disclosed herein.

[0007] In certain embodiments, the following components: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) Provided herein are empty lipid nanoparticle compositions comprising empty lipid nanoparticles comprising PEG-lipids; Empty lipid nanoparticle composition (a) is substantially free of payload; (b) having a pH of about 3 to about 5; (c) characterized by a zeta potential of about 35 mV or greater.

[0008] In certain embodiments, the following components: (i) ionized lipids, (ii) phospholipids, (iii) Structured lipids (iv) PEG-lipids, and (v) Provided herein are loaded lipid nanoparticle compositions comprising loaded lipid nanoparticles comprising a payload; The loaded lipid nanoparticle composition has a pH of about 4.5 to about 8.

[0009] In certain embodiments, provided herein are kits comprising a first container containing an empty lipid nanoparticle composition and a second container containing a solution comprising a therapeutic or prophylactic agent for combining with the empty lipid nanoparticle composition in the first container.

[0010] In certain embodiments, provided herein is a method of treating or preventing a disease in a patient, comprising administering to the patient a therapeutically effective amount of a loaded lipid nanoparticle composition disclosed herein.

[0011] Each limitation may encompass various embodiments. Thus, each limitation involving any one element or combination of elements is contemplated as being included in each aspect described. The invention is not limited in its application to the details of construction or to the arrangement of components set forth in the following description or illustrated in the drawings. Other embodiments and embodiments that are practiced or carried out in various ways are possible. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows a general process for preparing empty LNPs (eLNPs) involving nanoprecipitation at pH 4 followed by titration to pH 5.

[0013] [Diagram 2] The effect of pH and lipid solution concentration on the mean diameter (nm) of eLNPs is shown.

[0014] [Diagram 3] 1 shows the effect of buffer concentration and lipid solution concentration on the mean diameter (nm) of eLNPs.

[0015] [Figure 4] The effect of pH and buffer concentration on the mean diameter (nm) of eLNPs is shown.

[0016] [Diagram 5] 1 shows the effect of pH over time on the mean diameter (nm) of eLNPs.

[0017] [Figure 6] Zeta potential (mV) of eLNPs with 37.5 mM acetate buffer (left) or 75 mM acetate buffer (right) prepared with various concentrations of lipid solution (LSS) as a function of pH.

[0018] [Figure 7] Cryo-EM images of eLNPs precipitated at pH 4 (left) versus pH 5 (right) are shown.

[0019] [Figure 8] 1 shows a general process for preparing loaded LNPs (fLNPs) where encapsulation is performed at pH 5.

[0020] [Figure 9] FIG. 9 shows the average particle size and polydispersity index (PDI) of fLNPs prepared by the process of FIG.

[0021] [Figure 10] 1 shows capillary zone electrophoresis plots of eLNPs prepared at pH 4 and eLNPs prepared at pH 5 using acetate buffer at pH 5.

[0022] [Figure 11] FIG. 1 shows an alternative general process for preparing empty LNPs (eLNPs) where nanoprecipitation is carried out at pH 4.

[0023] [Figure 12] An alternative general process for preparing loaded LNPs (fLNPs) where encapsulation occurs at pH 4 to pH 6 is shown.

[0024] [Figure 13] The effect of pH on the mean diameter (nm) of fLNPs is shown.

[0025] [Figure 14] The effect of pH on the mean diameter (nm) of fLNPs prepared at pH 5 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Among other things, empty lipid nanoparticle (eLNP) compositions are provided, including their preparation and use, characterized by certain advantageous properties. In particular, some embodiments include empty lipid nanoparticle compositions having a substantially uniform morphology and a small average particle size with a size distribution, and having a relatively high zeta potential. The empty lipid nanoparticle compositions can be formed under conditions favoring a relative uniformity and small size that can remain stable for extended periods, even in the presence of ethanol, facilitating work-up and manipulation. In particular, the empty lipid nanoparticles can be formed under conditions of low pH, low ionic strength, and high buffer concentration. The combination of small size, uniform morphology, stability, and high zeta potential facilitates the use of the empty nanoparticle compositions in post-loading (PHL) of nucleic acids or other therapeutic agents, making them therapeutically effective loaded lipid nanoparticle (fLNP) compositions for delivery to cells of patients to treat or prevent disease.

[0027] Process for preparing empty lipid nanoparticle compositions The process for preparing the empty lipid nanoparticle composition may include nanoprecipitation of the empty lipid nanoparticles at low pH, low ionic strength, high buffer strength, or a combination thereof.

[0028] For example, some embodiments provide a process for preparing an empty lipid nanoparticle composition, comprising: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer having a pH of less than about 4.5.

[0029] In some embodiments, the aqueous buffer solution has a pH of about 3.5 to about 4.5. In other embodiments, the aqueous buffer solution has a pH of about 4.

[0030] The process of preparing the empty lipid nanoparticle composition may include precipitating nanoparticles at a relatively high buffer concentration, for example, a concentration high enough to dominate the buffering effect of lipids in the lipid solution. In some embodiments, the buffered aqueous solution has a buffer concentration of more than about 30 mM. In some embodiments, the buffered aqueous solution has a buffer concentration of more than about 40 mM. In some embodiments, the buffered aqueous solution has a buffer concentration of about 30 mM to about 100 mM. In some embodiments, the buffered aqueous solution has a buffer concentration of about 40 mM to about 75 mM. In another embodiment, the buffered aqueous solution has a buffer concentration of about 33 mM, about 37.5 mM, or about 45 mM.

[0031] In some embodiments, the aqueous buffer solution has a buffer concentration of about 45 mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 37.5 mM.

[0032] In some embodiments, the aqueous buffer used in the process of preparing the empty lipid nanoparticles has a pH lower than the pKa of the resulting empty lipid nanoparticles.

[0033] The process of preparing lipid nanoparticle composition may further comprise precipitating nanoparticles at a relatively low ionic strength.For example, the buffered aqueous solution may have an ionic strength of about 15 mM or less, about 10 mM or less, or about 5 mM or less.In some embodiments, the buffered aqueous solution has an ionic strength of about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.1 mM to about 5 mM.

[0034] Suitable buffers include those that maintain an acidic pH, such as a pH of 3 to 5. In some embodiments, the aqueous buffer may comprise an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the aqueous buffer comprises an acetate buffer or a citrate buffer. In another embodiment, the aqueous buffer is an acetate buffer, such as a sodium acetate buffer.

[0035] The process of preparing empty lipid nanoparticles may include precipitating nanoparticles under conditions that result in an empty lipid nanoparticle composition characterized by a relatively high zeta potential. In some embodiments, the process produces an empty lipid nanoparticle composition characterized by a zeta potential of about 35 mV or more, about 50 mV or more, or about 100 mV or more. In some embodiments, the process produces an empty lipid nanoparticle composition characterized by a zeta potential of about 35 mV to about 140 mV, about 50 mV to about 120 mV, or about 60 mV to about 100 mV. In some embodiments, the process produces empty lipid nanoparticle compositions characterized by a zeta potential that is at least about 25% of the maximum zeta potential achievable for the composition in the pH range of 3 to 6, at least about 33% of the maximum zeta potential achievable for the composition in the pH range of 3 to 6, at least about 50% of the maximum zeta potential achievable for the composition in the pH range of 3 to 6, at least about 66% of the maximum zeta potential achievable for the composition in the pH range of 3 to 6, or at least about 75% of the maximum zeta potential achievable for the composition in the pH range of 3 to 6.

[0036] Zeta potential measures the electrokinetic potential of a colloidal dispersion. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. Zeta potential can be measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential by the principle of "massively parallel phase analysis light scattering" or MP-PALS. This measurement is more sensitive and induces less stress than ISO method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. In some embodiments, the zeta potential of lipids in the empty lipid nanoparticle compositions described herein is measured using an instrument that utilizes the principle of MP-PALS.

[0037] The process may further use a lipid solution that is a composition that includes at least four lipid components: ionized lipid, phospholipid, structured lipid, and PEG-lipid. Any suitable concentration of lipid solution may be used. For example, the lipid solution may have a lipid concentration of about 5 to about 100 mg / mL, about 15 to about 35 mg / mL, about 20 to about 30 mg / mL, or about 24 mg / mL.

[0038] The lipid solution may further comprise an organic solvent, such as alcohol, for example, ethanol. The organic solvent can be present in an amount of about 1% to about 50% by volume, about 5% to about 40% by volume, or about 10% to about 33% by volume. In some embodiments, the solvent is 100% ethanol or more than 95% by volume.

[0039] In some embodiments, the lipid solution comprises about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, or about 40 mol% to about 50 mol% ionized lipid relative to total lipid.

[0040] In some embodiments, the lipid solution comprises about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10% to about 12 mol% phospholipids relative to total lipid.

[0041] In some embodiments, the lipid solution comprises about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 37% to about 42 mol% structured lipids relative to total lipids.

[0042] In some embodiments, the lipid solution comprises about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1 mol%, or about 0.25% to about 0.75 mol% PEG-lipid relative to the total lipid.

[0043] In some embodiments, the lipid solution contains, relative to total lipid, about 40 mol % to about 50 mol % ionized lipids; about 10 mol% to about 12 mol% phospholipids, about 37 mol % to about 42 mol % structured lipids, and Contains about 0.25 mol% to about 0.75 mol% PEG-lipid.

[0044] In some embodiments, the lipid solution contains, relative to total lipid, Approximately 49 mol% ionized lipids, about 11 mol% to about 12 mol% phospholipids, Approximately 39 mol% structural lipids, and Contains approximately 0.5 mol% PEG-lipid.

[0045] The lipid solution and the buffer solution are mixed to precipitate the lipid nanoparticles and prepare the empty lipid nanoparticle composition. Precipitation can be performed, for example, by ethanol precipitation using a high energy mixer (e.g., T-junction, confined impinging jet, microfluidic mixer, vortex mixer) that controllably introduces the lipid (in ethanol) into a suitable antisolvent (i.e., water) leading to liquid supersaturation and spontaneous precipitation into lipid particles. In some embodiments, mixing is performed using a multi-inlet vortex mixer. In some embodiments, mixing is performed using a microfluidic mixer as described in WO2014 / 172045. In some embodiments, the mixing step can be performed at ambient temperature or at a temperature, for example, less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

[0046] The precipitated empty lipid nanoparticles generally have a small average particle size, e.g., an average diameter of about 60 nm or less, about 50 nm or less, about 45 nm or less, about 30 nm or less, about 25 nm or less, or about 20 nm or less. In some embodiments, the empty lipid nanoparticles may have an average diameter of about 5 nm to about 30 nm or about 10 nm to about 20 nm. The average particle size can be measured, for example, by dynamic light scattering (DLS). Furthermore, the empty lipid nanoparticles may have a substantially uniform morphology. For example, the empty lipid particles may have a polydispersity index of about 1 or less, e.g., about 0.75 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, or 0.05 or less. See FIG. 7 for a comparison of cryo-EM images of empty lipid nanoparticles. In contrast to the image on the right, where the particles were prepared at pH 5, the image on the left shows particles with a uniform morphology prepared at pH 4 according to the present disclosure.

[0047] The precipitated empty lipid nanoparticles are substantially free of payload, where payload refers to any therapeutic or prophylactic agent, such as a polypeptide or nucleic acid, intended for delivery to a cell.

[0048] In some embodiments, the process results in a stable empty lipid nanoparticle composition. By "stable" it is meant that the empty lipid nanoparticles substantially maintain their size for an extended period of time. For example, the average diameter of the empty lipid nanoparticles increases less than about 150% over 25 hours, or increases less than about 100% over 25 hours. In some embodiments, the average diameter of the lipid nanoparticles remains less than 50 nm over 25 hours, or remains less than 40 nm over 25 hours. Stability is demonstrated in empty lipid nanoparticle compositions that include an organic solvent, such as alcohol (e.g., ethanol). In some embodiments, the empty lipid nanoparticles are stable in the presence of about 1% to about 30% by volume, about 10% to about 30% by volume, about 20% to about 30% by volume, or about 25% by volume of ethanol.

[0049] In some embodiments, the mean diameter of the lipid nanoparticles remains equal to or less than 50 nm for 25 hours at 25° C., or remains equal to or less than 40 nm for 25 hours at 25° C. In some embodiments, the mean diameter of the lipid nanoparticles remains equal to or less than 30 nm for at least 24 hours in the presence of 25% ethanol by volume ... at 25° C.

[0050] In some embodiments, the empty lipid nanoparticle composition is in a storage solution. In some embodiments, the storage solution comprises a buffer. In some embodiments, the buffer concentration is about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 to about 20 mM, about 1 to about 10 mM, or about 5 mM. In some embodiments, the buffer in the storage solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In some embodiments, the buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the cryoprotectant solution is about 3 to about 8, about 4 to about 7, about 4, about 5, about 6, about 7, or about 8.

[0051] In some embodiments, the preservation solution includes a cryoprotectant. In some embodiments, the cryoprotectant includes one or more cryoprotectants, such as a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-surfactant sulfonate, a glycerol ... Phosphatase inhibitors (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso- In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is sodium chloride. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is sodium chloride. In some embodiments, the cryoprotectant is sucrose and sodium chloride.

[0052] In some embodiments, the empty lipid nanoparticle concentration in the storage solution is from about 5 to about 100 mg / mL, from about 15 to about 75 mg / mL, or from about 20 to about 60 mg / mL.

[0053] In some embodiments, the storage solution containing the empty lipid nanoparticles is maintained at about 15° C. to about 25° C., about 15° C. to about 20° C., or about 18° C. to about 20° C. In some embodiments, the storage solution containing the empty lipid nanoparticles is maintained at about 1° C. to about 10° C., about 2° C. to about 9° C., or about 3° C. to about 7° C.

[0054] In some embodiments, the average diameter of the empty lipid nanoparticles in the storage solution remains equal to or less than 30 nm for at least 4 months at 5°C.

[0055] The process of preparing the empty lipid nanoparticle composition may further comprise one or more additional steps selected from the following: diluting the composition with a dilution buffer; adjusting the pH of the composition to a pH of about 5 to about 6; filtering the composition; concentrating the composition; exchanging the buffer solution of the composition; and Adding a cryoprotectant to the composition.

[0056] In some embodiments, the process of preparing empty lipid nanoparticle composition may further include one, two, three, four, five, or all of the steps listed above. Some steps may be repeated. The steps may be, but need not be, performed in the order listed. Each step refers to an act related to the composition obtained from the preceding step. For example, if the process includes a step of exchanging the buffer of the composition, the buffer exchange is performed on the composition obtained from the preceding step, which may be any of the steps listed above.

[0057] In some embodiments, the process includes diluting the composition with a dilution buffer. The dilution step can be useful for reducing the proportion of organic solvent in the empty lipid nanoparticle composition. The dilution buffer can be an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 30 mM to about 75 mM, about 30 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the dilution buffer includes an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the dilution buffer comprises an acetate buffer or a citrate buffer. In another embodiment, the dilution buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the dilution buffer is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, or about 6. In some embodiments, the dilution buffer comprises the same buffer as in the aqueous buffer solution used to precipitate the empty lipid nanoparticles. In some embodiments, the dilution buffer has a pH that is the same as or higher than the pH of the aqueous buffer solution used to precipitate the empty lipid nanoparticles.

[0058] For example, diluting a composition with a dilution buffer may correspond to the ILD buffer step in FIG.

[0059] In some embodiments, the process includes adjusting the pH of the composition to a pH of about 5 to about 6. For example, if the empty lipid nanoparticle composition has undergone nanoprecipitation at pH 4, the pH of the composition can be increased by adding a higher pH buffer. In some embodiments, the pH is adjusted to about pH 5.

[0060] For example, adjusting the pH of the composition to a pH of about 5 to about 6 may correspond to the ILD buffer step in FIG.

[0061] In some embodiments, the process does not include adjusting the pH of the composition. For example, if the empty lipid nanoparticle composition has undergone nanoprecipitation at pH 4, the pH of the composition is maintained at about 4.

[0062] In some embodiments, the buffer for pH adjustment may be an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is stable at about 30 mM to about 75 mM, about 30 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the buffer for pH adjustment comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer for pH adjustment comprises an acetate buffer or a citrate buffer. In another embodiment, the buffer for pH adjustment is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the buffer solution for pH adjustment is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6. In some embodiments, the buffer used to adjust the pH can also be a dilution buffer.

[0063] In some embodiments, the process includes adding a cryoprotectant to the composition. In some embodiments, the cryoprotectant is one or more cryoprotectants, such as a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-surfactant sulfonate, a glycerol ... Phosphatase inhibitors (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso- In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant is sucrose.

[0064] The cryoprotectant may be added to the empty lipid nanoparticle composition by the addition of an aqueous cryoprotectant solution, which may include an aqueous buffer having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 to about 20 mM, about 1 to about 10 mM, or about 5 mM. In some embodiments, the buffer in the cryoprotectant solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In another embodiment, the buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the cryoprotectant solution is about 3 to about 6, about 4 to about 6, about 4, about 5, or about 6.

[0065] In some embodiments, the buffer concentration is about 20 mM to about 60 mM, about 25 mM to about 55 mM, about 30 mM to about 50 mM, or about 30 mM to about 40 mM. In some embodiments, the buffer concentration is about 30 mM to about 38 mM, about 33 mM to about 38 mM, about 35 mM to about 38 mM, or about 37 mM to about 38 mM. In some embodiments, the buffer concentration is about 37 mM to about 44 mM, about 37 mM to about 42 mM, or about 37 mM to about 40 mM. In some embodiments, the buffer concentration is about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, or about 40 mM. In some embodiments, the buffer concentration is about 37.5 mM.

[0066] In some embodiments, the cryoprotectant solution comprises about 40% w / v to about 90% w / v, about 50% w / v to about 85% w / v, about 60% w / v to about 80% w / v, or about 70% w / v sucrose.

[0067] For example, adding a cryoprotectant to a composition may correspond to the sucrose addition step in FIGS.

[0068] In some embodiments, the process includes one or more of the steps of filtering the composition, concentrating the composition, and exchanging a buffer solution for the composition. The filtering, concentrating, and buffer exchange steps can be performed using tangential flow filtration (TFF).

[0069] For example, filtering a composition, concentrating a composition, and buffer exchanging a composition can correspond to the TFF steps of FIGS.

[0070] In some embodiments, the filtering step can remove organic solvents (e.g., alcohols such as ethanol) and other undesirable components from the lipid nanoparticle composition.

[0071] In some embodiments, buffer exchange can change the composition of the empty lipid nanoparticle composition by increasing or decreasing the buffer concentration, changing the buffer composition, removing or reducing the amount of organic solvent, or changing the pH. In some embodiments, the buffer exchange step includes decreasing the buffer concentration, for example, from about 1 mM to about 10 mM, from about 2 mM to about 8 mM, from about 4 mM to about 6 mM, or to about 5 mM. In some embodiments, the buffer exchange step includes removing or reducing the amount of organic solvent.

[0072] In some embodiments, the concentrating step can increase the concentration of empty lipid nanoparticles in the composition.

[0073] In some embodiments, the process includes at least the step of adjusting the pH of the composition to a pH of about 5 to about 6.

[0074] In some embodiments, the process includes at least the step of adjusting the pH of the composition to a pH of about 4.5 to about 6.

[0075] In some embodiments, the process comprises at least two steps: adjusting the pH of the composition to a pH of about 5 to about 6, and adding a cryoprotectant to the composition.

[0076] In some embodiments, the process comprises at least two steps: adjusting the pH of the composition to a pH of about 4.5 to about 6, and adding a cryoprotectant to the composition.

[0077] In some embodiments, the process includes at least one step comprising TFF, in which the composition undergoes filtration, buffer exchange, concentration, or a combination thereof.

[0078] In some embodiments, the process includes diluting the composition.

[0079] In some embodiments, the composition may be diluted with a dilution buffer. The dilution buffer may be an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 30 mM to about 75 mM, about 30 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the dilution buffer comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the dilution buffer comprises an acetate buffer or a citrate buffer. In another embodiment, the dilution buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the dilution buffer is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, or about 6. In some embodiments, the dilution buffer comprises the same buffer as that in the aqueous buffer solution used to precipitate the empty lipid nanoparticles.

[0080] In some embodiments, the composition is diluted with acetic acid solution, sodium acetate solution, citric acid solution, sodium citrate solution, phosphoric acid solution, or sodium phosphate solution. In some embodiments, diluting the composition increases the pH of the composition. In some embodiments, diluting the composition decreases the pH of the composition. In some embodiments, the pH of the composition after the composition is diluted is about 4, about 4.5, about 5, about 5.5, or about 6.

[0081] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) optionally adjusting the pH of the solution from the previous step by addition of a pH adjusting buffer having a pH of about 5 to about 6; (c) optionally filtering and reducing the buffer concentration of the solution from the previous step; (d) optionally adding sucrose to the solution from the previous step; (e) optionally, diluting the solution from the previous step.

[0082] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) adjusting the pH of the solution from the previous step by the addition of a pH adjusting buffer having a pH of about 5 to about 6; (c) optionally filtering and reducing the buffer concentration of the solution from the previous step; (d) adding sucrose to the solution from the previous step; (e) optionally, diluting the solution from the previous step.

[0083] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) optionally adding sucrose to the solution from the previous step; (c) optionally, diluting the solution from the previous step.

[0084] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) adding sucrose to the solution from the previous step; (c) optionally, diluting the solution from the previous step.

[0085] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) adjusting the pH of the composition to a pH of about 5 to about 6; (c) filtering the composition; (d) concentrating the composition; and (e) buffer exchanging the composition; and (f) adding a cryoprotectant to the composition.

[0086] In some embodiments, the empty lipid nanoparticle composition comprises: (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid mixing with an aqueous buffer solution having a pH of about 4; (b) diluting the composition with a dilution buffer; (c) filtering the composition; (d) concentrating the composition; and (e) buffer exchanging the composition; and (f) adding a cryoprotectant to the composition.

[0087] Some embodiments include an empty lipid nanoparticle composition prepared by any of the processes described herein.

[0088] Uses of Empty Lipid Nanoparticle Compositions The empty lipid nanoparticle composition can be used to prepare a filled lipid nanoparticle (fLNP) composition by combining the empty lipid nanoparticle composition with a payload.

[0089] Some embodiments include a process for preparing a filled lipid nanoparticle composition comprising combining an empty lipid nanoparticle composition, such as one prepared by any of the processes described herein, with a payload to form a filled lipid nanoparticle composition.

[0090] Some embodiments include (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid with an aqueous buffer solution having a pH of less than about 4.5 to obtain a blank lipid nanoparticle composition; (b) combining the empty lipid nanoparticle composition with a payload to form a filled lipid nanoparticle composition.

[0091] In some embodiments, the combining is performed at a pH of about 4.5 to about 5.5. In some embodiments, the combining is performed at a pH of about 5. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 4.5 to about 5.5 prior to combining the empty lipid nanoparticle composition with the payload. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 5 prior to combining the empty lipid nanoparticle composition with the payload.

[0092] In some embodiments, the combining is performed at a pH of about 4 to about 6. In some embodiments, the combining is performed at a pH of about 4, about 4.5, about 5, about 5.5, or about 6. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 4 to about 6 prior to combining the empty lipid nanoparticle composition with the payload. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 4, about 4.5, about 5, about 5.5, or about 6 prior to combining the empty lipid nanoparticle composition with the payload.

[0093] In some embodiments, the payload is a nucleic acid. The nucleic acid payload may be provided as a nucleic acid solution comprising (i) a nucleic acid, such as DNA or RNA (e.g., mRNA), and (ii) a buffer capable of maintaining an acidic pH, such as a pH of about 3 to about 6, about 4 to about 6, or about 5 to about 6. In some embodiments, the pH of the nucleic acid solution is about 5.

[0094] In some embodiments, the pH of the nucleic acid solution is about 4, about 4.5, about 5, about 5.5, or about 6.

[0095] In some embodiments, the buffer of the nucleic acid solution is an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In another embodiment, the buffer is an acetate buffer, such as a sodium acetate buffer. The buffer concentration of the nucleic acid solution may be about 5 mM to about 140 mM. In some embodiments, the buffer concentration is about 20 mM to about 100 mM, about 30 mM to about 70 mM, or about 40 mM to about 50 mM. In some embodiments, the buffer concentration is about 42.5 mM.

[0096] In some embodiments, the buffer concentration is about 20 mM to about 60 mM, about 25 mM to about 55 mM, about 30 mM to about 50 mM, or about 30 mM to about 40 mM. In some embodiments, the buffer concentration is about 30 mM to about 38 mM, about 33 mM to about 38 mM, about 35 mM to about 38 mM, or about 37 mM to about 38 mM. In some embodiments, the buffer concentration is about 37 mM to about 44 mM, about 37 mM to about 42 mM, or about 37 mM to about 40 mM. In some embodiments, the buffer concentration is about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, or about 40 mM. In some embodiments, the buffer concentration is about 37.5 mM.

[0097] The nucleic acid solution may contain a nucleic acid concentration of about 0.05 to about 5.0 mg / mL, 0.05 to about 2.0 mg / mL, about 0.05 to about 1.0 mg / mL, about 0.1 to about 0.5 mg / mL, or about 0.2 to about 0.3 mg / mL. In some embodiments, the nucleic acid concentration is about 0.25 mg / mL.

[0098] In some embodiments, the nucleic acid concentration is about 0.2 mg / mL to about 2.0 mg / mL, about 0.4 mg / mL to about 1.8 mg / mL, about 0.6 mg / mL to about 1.4 mg / mL, or about 0.8 mg / mL to about 1.2 mg / mL. In some embodiments, the nucleic acid concentration is about 0.5 mg / mL, about 0.7 mg / mL, about 1.3 mg / mL, or about 1.5 mg / mL. In some embodiments, the nucleic acid concentration is about 1.0 mg / mL. In some embodiments, the nucleic acid concentration is about 0.8 mg / mL to about 2.6 mg / mL, about 1.0 mg / mL to about 2.4 mg / mL, about 1.2 mg / mL to about 2.0 mg / mL, or about 1.4 mg / mL to about 1.8 mg / mL. In some embodiments, the nucleic acid concentration is about 1.1 mg / mL, about 1.3 mg / mL, about 1.9 mg / mL, or about 2.1 mg / mL. In some embodiments, the nucleic acid concentration is about 1.6 mg / mL.

[0099] In some embodiments, the nucleic acid concentration is about 0.05 mg / mL to about 0.9 mg / mL, about 0.07 mg / mL to about 0.7 mg / mL, about 0.09 mg / mL to about 0.5 mg / mL, or about 0.2 mg / mL to about 0.3 mg / mL. In some embodiments, the nucleic acid concentration is about 0.15 mg / mL, about 0.25 mg / mL, about 0.35 mg / mL, or about 0.45 mg / mL. In some embodiments, the nucleic acid concentration is about 0.25 mg / mL.

[0100] In some embodiments, the nucleic acid concentration is about 0.08 mg / mL to about 1.3 mg / mL, about 0.1 mg / mL to about 1.1 mg / mL, about 0.3 mg / mL to about 0.9 mg / mL, or about 0.5 mg / mL to about 0.7 mg / mL. In some embodiments, the nucleic acid concentration is about 0.46 mg / mL, about 0.56 mg / mL, about 0.66 mg / mL, or about 0.76 mg / mL. In some embodiments, the nucleic acid concentration is about 0.56 mg / mL.

[0101] The empty lipid nanoparticles are post-loaded with nucleic acid by mixing the empty lipid nanoparticle composition and the nucleic acid solution. A high energy mixer (e.g., T-junction, confined impinging jet, microfluidic mixer, vortex mixer) may be used. In some embodiments, the mixing is performed using a multi-inlet vortex mixer. In some embodiments, the mixing is performed using a microfluidic mixer as described in WO2014 / 172045. The mixing step can be performed at ambient temperature or at a temperature, for example, less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

[0102] The encapsulation efficiency for the empty lipid nanoparticle compositions disclosed herein is advantageously high. Encapsulation efficiency (EE) indicates the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with lipid nanoparticles after preparation relative to the amount initially prepared. Encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent (e.g., payload) in a solution containing lipid nanoparticles before and after disintegrating the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins may be used to measure the amount of therapeutic and / or prophylactic agent (e.g., RNA) free in solution. Fluorescence may be used to measure the amount of therapeutic and / or prophylactic agent (e.g., RNA) free in solution. The encapsulation efficiency of the therapeutic and / or prophylactic agent for the lipid nanoparticle compositions described herein is at least about 50%, e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%. In some embodiments, the encapsulation efficiency can be about 90% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more.

[0103] In some embodiments, the process of preparing a loaded lipid nanoparticle composition further comprises one or more additional steps selected from the following: diluting the composition with a dilution buffer; adjusting the pH of the composition to a pH of about 7 to about 8; filtering the composition; concentrating the composition; exchanging the buffer solution of the composition; adding one or more surfactants to the composition; adding a cryoprotectant to the composition; and Adding an osmolality adjusting agent to the composition.

[0104] In some embodiments, the process of preparing loaded lipid nanoparticle composition may further include 1, 2, 3, 4, 5, 6, 7, or all of the steps listed above. Some steps may be repeated. The steps may be, but need not be, performed in the order listed. Each step refers to an act related to the composition obtained from the preceding step. For example, if the process includes a step of adding one or more surfactants to the composition, the surfactants are added to the composition obtained from the preceding step, which may be any of the steps listed above.

[0105] In some embodiments, one or more additional steps is adjusting the pH of the composition to a pH of about 7 to about 8. In some embodiments, the pH is adjusted to a pH of about 7.5.

[0106] In some embodiments, the pH is adjusted to a pH of about 7.2.

[0107] In some embodiments, the pH is adjusted by adding a neutralization buffer. For example, adding a neutralization buffer may correspond to the neutralization buffer step of FIG. 8 or FIG. 12. In some embodiments, the neutralization buffer comprises an aqueous buffer having a buffer concentration of about 1 mM to about 200 mM, about 10 mM to about 190 mM, about 20 mM to about 190 mM, about 30 mM to about 180 mM, about 40 mM to about 170 mM, about 50 mM to about 160 mM, about 60 mM to about 150 mM, about 70 mM to about 140 mM, about 80 mM to about 130 mM, about 90 mM to about 130 mM, or about 110 mM to about 125 mM. In some embodiments, the buffer concentration is about 110 mM, about 120 mM, or about 130 mM. In some embodiments, the neutralization buffer comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is a Tris buffer. In some embodiments, the pH of the neutralization buffer is about 7.0 to about 8.5, about 7.4 to about 8.5, about 7.6 to about 8.5, about 7.8 to about 8.5, or about 8.0 to about 8.4. In some embodiments, the pH of the neutralization buffer is about 8.0 to about 8.15, about 8.15 to about 8.25, or about 8.25 to about 8.35. In some embodiments, the pH of the neutralization buffer is about 8.12, about 8.2, or about 8.3. In some embodiments, the neutralization buffer comprises sucrose. In some embodiments, the neutralization buffer comprises about 12% w / v to about 22% w / v, about 14% w / v to about 20% w / v, about 16% w / v to about 18% w / v, or about 17% w / v sucrose.

[0108] In some embodiments, one or more additional steps are to add surfactant to the composition.Surfactants can include, but are not limited to, PEG derivatives (e.g., PEG-DMG), lipid amines (e.g., sterol amines and related), anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromexin, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, tiopronin, gelsolin, thymosin, β4, dornase alpha, neltenexin, and erdostein), and DNase (e.g., rhDNase). The surfactant may be disposed within and / or on the surface of the nanoparticle (eg, by coating, adsorption, covalent bonding, or other process).

[0109] For example, adding an additional surfactant to the composition may correspond to the PI buffer step in FIG.

[0110] In some embodiments, one or more additional steps is adding an osmotic modifier to the composition. The osmotic modifier may be a salt or a sugar. In some embodiments, the osmotic modifier is a sugar. The sugar may be selected from, but is not limited to, glucose, fructose, galactose, sucrose, lactose, maltose, and dextrose. In some embodiments, the osmotic modifier is a salt. The salt may be an inorganic salt, such as sodium chloride, potassium chloride, calcium chloride, or magnesium chloride. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the salt is 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt. The salt may be provided as a salt solution having a salt concentration of about 100 to about 500 mM, about 200 to about 400 mM, about 250 to about 350 mM, or about 300 mM. The pH of the salt solution may be about 7 to about 8. The salt solution may further comprise a buffer, including, for example, acetate, citrate, phosphate, or Tris buffer. The buffer concentration may be, for example, about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM.

[0111] For example, adding an osmolality adjusting agent to a composition may correspond to the salt addition step in FIG.

[0112] The cryoprotectant may be added to the loaded particle composition by the addition of an aqueous cryoprotectant solution, which may comprise an aqueous buffer having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 to about 20 mM, about 1 to about 10 mM, or about 5 mM. In some embodiments, the buffer in the cryoprotectant solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In another embodiment, the buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the cryoprotectant solution is about 7 to about 8, e.g., about 7.5. In some embodiments, the cryoprotectant solution comprises about 40% to about 90%, about 50% to about 85%, about 60% to about 80%, or about 70% sucrose by weight.

[0113] For example, adding a cryoprotectant to a composition may correspond to the fill and complete step of FIG.

[0114] In some embodiments, the process includes diluting the composition with a dilution buffer. The dilution buffer may be an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 30 mM to about 75 mM, about 30 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the dilution buffer comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the dilution buffer comprises an acetate buffer or a citrate buffer. In another embodiment, the dilution buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the dilution buffer is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, or about 6. In some embodiments, the dilution buffer comprises the same buffer as in the buffered aqueous solution used during the combination of the blank lipid nanoparticle composition with the nucleic acid solution.

[0115] In some embodiments, the process includes one or more of the steps of filtering the composition, concentrating the composition, and exchanging the buffer of the composition. The filtering, concentrating, and buffer exchange steps can be performed using tangential flow filtration (TFF). Residual organic solvent can be removed by the filtering step.

[0116] For example, filtering a composition, concentrating a composition, or buffer exchanging a composition can correspond to the TFF filtration step of FIG.

[0117] In some embodiments, buffer exchange can change the composition of the loaded lipid nanoparticle composition by increasing or decreasing the buffer concentration, changing the buffer composition, or changing the pH.

[0118] In some embodiments, the concentrating step can increase the concentration of loaded lipid nanoparticles in the composition.

[0119] In some embodiments, the process of preparing a loaded lipid nanoparticle composition further comprises adjusting the pH of the composition to at least a pH of about 7 to about 8 (e.g., about pH 7.5) and adding an osmolality adjusting agent (e.g., an inorganic salt) to the composition.

[0120] In some embodiments, the process of preparing a loaded lipid nanoparticle composition further comprises adjusting the pH of the composition to at least a pH of about 7 to about 8 (e.g., about pH 7.5), adding a surfactant to the composition, and adding an osmotic agent (e.g., an inorganic salt) to the composition.

[0121] The empty lipid nanoparticle composition is mixed with the nucleic acid solution to obtain a filled lipid nanoparticle composition. The mixing may be performed by a high energy mixer (e.g., a T-junction, a confined impinging jet, a microfluidic mixer, a vortex mixer). In some embodiments, the mixing is performed using a multi-inlet vortex mixer. In some embodiments, the mixing is performed using a microfluidic mixer as described in WO2014 / 172045. The mixing step can be performed at ambient temperature or at a temperature, for example, less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

[0122] Generally speaking, the loaded lipid nanoparticle composition comprises nanoparticles having a larger average diameter than the starting empty particles. For example, the loaded particles may have an average diameter of less than about 160 nm, less than about 150 nm, less than about 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, or less than about 70 nm. In some embodiments, the loaded lipid nanoparticle composition comprises particles having an average diameter of about 50 to about 160 nm, about 50 to about 140 nm, about 50 to about 120 nm, about 50 to about 100 nm, about 60 to about 100 nm, about 70 to about 90 nm, about 75 to about 90, or 75 to about 85 nm. Furthermore, the loaded lipid nanoparticle composition is characterized by a polydispersity index (PDI). For example, the polydispersity index (PDI) for the loaded lipid nanoparticle composition disclosed herein may be about 0.12 to about 0.25.

[0123] In some embodiments, the loaded lipid nanoparticle composition is in a storage solution. In some embodiments, the storage solution comprises a buffer. In some embodiments, the buffer concentration is about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 to about 20 mM, about 1 to about 10 mM, or about 5 mM. In some embodiments, the buffer in the storage solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In some embodiments, the buffer is an acetate buffer, such as sodium acetate. In some embodiments, the buffer is acetate buffer or Tris buffer. In some embodiments, the pH of the cryoprotectant solution is about 3 to about 8, about 4 to about 7, about 4, about 5, about 6, about 7, about 7.5, or about 8.

[0124] In some embodiments, the preservation solution includes a cryoprotectant. In some embodiments, the cryoprotectant includes one or more cryoprotectants, such as a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-surfactant sulfonate, a glycerol ... Phosphatase inhibitors (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG200), PEG400, PEG600, PEG1000, PEG3350, PEG4000, PEG8000, PEG10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG600, mPEG2000, mPEG3350, mPEG4000, mPEG5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K15), pentaerythritol propoxylate, or polypropylene glycol P400), organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso- In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is sodium chloride. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is sodium chloride. In some embodiments, the cryoprotectant is sucrose and sodium chloride.

[0125] In some embodiments, the loaded lipid nanoparticle concentration in the storage solution is from 0.5 to about 10 mg / mL, from about 1 to about 5 mg / mL, or from about 1 to about 3 mg / mL.

[0126] In some embodiments, the storage solution containing the loaded lipid nanoparticles is maintained at about 15° C. to about 25° C., about 15° C. to about 20° C., or about 18° C. to about 20° C. In some embodiments, the storage solution containing the loaded lipid nanoparticles is maintained at about 1° C. to about 10° C., about 2° C. to about 9° C., or about 3° C. to about 7° C.

[0127] In some embodiments, the process for preparing a loaded lipid nanoparticle composition comprises: (c) adjusting the pH of the composition to a pH of about 7 to about 8; (d) adding one or more surfactants to the composition; (e) concentrating the composition; and (f) adding an inorganic salt to the composition; (g) diluting the composition.

[0128] Some embodiments include (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid with an aqueous buffer solution having a pH of less than about 4.5 to obtain a blank lipid nanoparticle composition; (b) combining the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition; (c) adjusting the pH of the composition to a pH of about 7 to about 8; (d) adding one or more surfactants to the composition; (e) filtering the composition; (f) concentrating the composition; and (g) buffer exchanging the composition; and (h) adding an osmolality adjusting agent to the composition; The present invention includes a process for preparing a loaded lipid nanoparticle composition comprising: (i) adding a cryoprotectant to the composition.

[0129] Some embodiments include (a) (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) A lipid solution containing PEG-lipid with an aqueous buffer solution having a pH of less than about 4.5 to obtain a blank lipid nanoparticle composition; (b) combining the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition; (c) adjusting the pH of the composition to a pH of about 7 to about 8; (d) adding one or more surfactants to the composition.

[0130] Some embodiments include a loaded lipid nanoparticle composition prepared by any of the processes described herein for preparing a loaded lipid nanoparticle composition.

[0131] Lipid Nanoparticle Compositions Empty lipid nanoparticle compositions are also provided, including any of a number of other components, such as water, organic solvents, buffers, cryoprotectants, pharmaceutical additives, or combinations thereof. The empty lipid nanoparticles are suitable for preparing loaded or filled lipid nanoparticle compositions for therapeutic or prophylactic use. The empty lipid nanoparticle compositions may be provided in liquid form, where water and / or organic solvents are present in the composition and the particles are suspended or otherwise present in a liquid medium. The empty lipid nanoparticle compositions may also be provided in solid form, such as frozen or lyophilized form.

[0132] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) an empty lipid nanoparticle composition comprising empty lipid nanoparticles comprising PEG-lipids; The empty lipid nanoparticle composition comprises (a) is substantially free of payload; (b) having a pH of about 3 to about 5; (c) characterized by a zeta potential of about 35 mV or greater.

[0133] Empty lipid nanoparticle compositions are substantially free of payload, e.g., substantially free of any therapeutic or prophylactic proteins or nucleic acids, thereby making the compositions useful for preparing loaded or filled lipid nanoparticles that contain payload.

[0134] The empty lipid nanoparticle composition may be characterized by a relatively high zeta potential of about 35 mV or more. In some embodiments, the empty lipid nanoparticle composition is characterized by a zeta potential of about 50 mV or more, or about 100 mV or more. In other embodiments, the empty lipid nanoparticle composition is characterized by a zeta potential of about 35 mV to about 140 mV, about 50 mV to about 120 mV, or about 60 mV to about 100 mV. In some embodiments, the empty lipid nanoparticle composition is characterized by a zeta potential that is at least about 25% of the maximum zeta potential achievable for the composition in a pH range of 3 to 6, at least about 33% of the maximum zeta potential achievable for the composition in a pH range of 3 to 6, at least about 50% of the maximum zeta potential achievable for the composition in a pH range of 3 to 6, at least about 66% of the maximum zeta potential achievable for the composition in a pH range of 3 to 6, or at least about 75% of the maximum zeta potential achievable for the composition in a pH range of 3 to 6.

[0135] The empty lipid nanoparticle composition may be characterized as having an acidic pH, for example, of about 3 to about 5. In some embodiments, the composition has a pH of about 3.5 to about 4.5. In another embodiment, the composition has a pH of about 4. In another embodiment, the composition has a pH of about 5.

[0136] The empty lipid nanoparticle composition may be characterized as having empty lipid nanoparticles with an average diameter of less than about 30 nm, less than about 25 nm, or less than about 20 nm. In some embodiments, the empty lipid nanoparticles of the composition have an average diameter of about 5 nm to about 20 nm, about 8 nm to about 20 nm, or about 10 nm to about 20 nm.

[0137] The empty lipid nanoparticle composition may be further characterized by a polydispersity index (PDI), which can be used to indicate the homogeneity, e.g., particle size distribution, of the lipid nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The empty lipid nanoparticle composition described herein may have a polydispersity index of about 0 to about 0.25, about 0.10 to about 0.25, about 0.15 to about 0.25, or about 0.2 to about 0.25.

[0138] In some embodiments, the empty lipid nanoparticle composition has a concentration of empty lipid nanoparticles of about 1 to about 100 mg / mL, about 25 to about 75 mg / mL, about 40 to about 60 mg / mL, or about 50 mg / mL.

[0139] In some embodiments, the empty lipid nanoparticle composition comprises a buffer.For example, the composition may comprise about 1 to about 100 mM buffer, about 1 to about 10 mM buffer, or about 5 mM buffer.Suitable buffers are any that can maintain an acidic pH at relatively low ionic strength.Exemplary buffers include acetate buffer, citrate buffer, phosphate buffer, Tris buffer, or combinations thereof.

[0140] The empty lipid nanoparticle composition may further comprise a cryoprotectant, such as, for example, any of those described herein. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the empty lipid nanoparticle composition may comprise about 1 to about 50% w / v, about 10 to about 30% w / v, or about 20% w / v of sucrose (or other cryoprotectant). In another embodiment, the empty lipid nanoparticle composition may comprise about 1 to about 15% w / v, about 5 to about 10% w / v, about 7 to about 8% w / v, or about 7.5% w / v of sucrose (or other cryoprotectant).

[0141] In some embodiments, the empty lipid nanoparticle composition of the present invention may further comprise an organic solvent. The organic solvent is generally miscible with water. Exemplary organic solvents include alcohols such as ethanol. In some embodiments, the organic solvent is present in an amount of about 25% or less by volume. In some embodiments, the empty lipid nanoparticle composition comprises about 25% ethanol by volume. In some embodiments, the empty lipid nanoparticle composition comprises 0 to about 25%, about 0 to about 10%, or 0 to about 5% organic solvent. In some embodiments, the empty lipid nanoparticle composition is substantially free of organic solvent.

[0142] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) an empty lipid nanoparticle composition comprising about 1 to about 100 mg / mL of empty lipid nanoparticles comprising PEG-lipids; The empty lipid nanoparticle composition comprises (a) is substantially free of payload; (b) having a pH of about 4 to about 5; (c) characterized by a zeta potential of about 35 mV or greater; (d) further comprising about 1 mM to about 100 mM of a buffer selected from an acetate buffer or a citrate buffer; (e) further comprising about 1 to about 50% w / v cryoprotectant.

[0143] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) an empty lipid nanoparticle composition comprising about 25 to about 75 mg / mL of empty lipid nanoparticles comprising PEG-lipids; The empty lipid nanoparticle composition comprises (a) is substantially free of payload; (b) having a pH of about 5; (c) characterized by a zeta potential of about 35 mV or greater; (d) further comprising about 1 mM to about 10 mM acetate buffer; (e) further containing about 10 to about 30% w / v sucrose.

[0144] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structural lipids, and (iv) an empty lipid nanoparticle composition comprising about 50 mg / mL of empty lipid nanoparticles comprising PEG-lipids; The empty lipid nanoparticle composition comprises (a) is substantially free of payload; (b) having a pH of about 5; (c) characterized by a zeta potential of about 35 mV or greater; (d) further comprising about 5 mM acetate buffer; (e) further containing about 20% w / v sucrose.

[0145] Some embodiments include a loaded lipid nanoparticle composition comprising loaded lipid nanoparticles and any of a number of other components, such as water, an organic solvent, a buffer, a cryoprotectant, or a combination thereof. The loaded lipid nanoparticles are generally suitable for therapeutic or prophylactic use in patients. The loaded lipid nanoparticle composition may be provided in a liquid form, where water and / or an organic solvent is present in the composition and the particles are suspended or otherwise present in a liquid medium. The loaded lipid nanoparticle composition may also be provided in a solid form, such as a frozen or lyophilized form.

[0146] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, and (v) a loaded lipid nanoparticle composition comprising loaded lipid nanoparticles comprising a payload, The loaded lipid nanoparticle composition has a pH of about 4.5 to about 8.

[0147] The filled lipid nanoparticle composition can be prepared by filling the empty lipid nanoparticle composition described herein. The filled lipid nanoparticle composition may have a pH of about 5 to about 8. In some embodiments, for example, the filled lipid nanoparticle composition obtained directly from filling may have a pH of about 5. In some embodiments, for example, when the filled lipid nanoparticle composition is neutralized, it may have a pH of about 7 to about 8, for example, about 7.5.

[0148] In some embodiments, the loaded lipid nanoparticle composition has a payload concentration of about 0.1 to about 10 mg / mL, about 0.5 to about 5 mg / mL, about 1 to about 2 mg / mL, about 2 mg / mL, or about 1 mg / mL.

[0149] In some embodiments, the loaded lipid nanoparticle composition further comprises a cryoprotectant, such as any cryoprotectant described herein. The loaded lipid nanoparticle composition may comprise a cryoprotectant in an amount of about 0.1% w / v to about 10% w / v, about 1% w / v to about 5% w / v, or about 3% w / v to about 4% w / v. In some embodiments, the cryoprotectant is sucrose.

[0150] In some embodiments, the loaded lipid nanoparticle composition further comprises an inorganic salt, such as any inorganic salt described herein.In some embodiments, the loaded lipid nanoparticle composition comprises about 5 mM to about 150 mM, about 10 mM to about 100 mM, about 50 mM to about 90 mM, or about 70 mM.In some embodiments, the inorganic salt is NaCl.

[0151] In some embodiments, the loaded lipid nanoparticle composition further comprises a buffer. Exemplary buffers include acetate buffer, citrate buffer, phosphate buffer, Tris buffer, or a combination thereof. In some embodiments, the loaded lipid nanoparticle composition comprises about 5 mM to about 100 mM buffer, about 7.5 mM to about 75 mM buffer, about 10 mM to about 50 mM buffer, or about 30 mM to about 50 mM buffer. In some embodiments, the buffer comprises acetate buffer or Tris buffer, or a combination thereof. In another embodiment, the buffer comprises acetate buffer and Tris buffer.

[0152] The loaded lipid nanoparticle composition may be further characterized by its average diameter. The loaded lipid nanoparticle may have an average diameter larger than the starting empty particle. For example, the loaded particles may have an average diameter of less than about 160 nm, less than about 150 nm, less than about 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, or less than about 70 nm. In some embodiments, the loaded lipid nanoparticle composition comprises particles having an average diameter of about 50 to about 160 nm, about 50 to about 140 nm, about 50 to about 120 nm, about 50 to about 100 nm, about 60 to about 100 nm, about 70 to about 90 nm, about 75 to about 90, or 75 to about 85 nm.

[0153] The loaded lipid nanoparticle compositions may be further characterized by a polydispersity index (PDI). The loaded lipid nanoparticle compositions described herein may have a polydispersity index of about 0 to about 0.25, about 0.10 to about 0.25, about 0.15 to about 0.25, or about 0.2 to about 0.25.

[0154] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, and (v) a loaded lipid nanoparticle composition comprising a payload, The loaded lipid nanoparticle composition comprises (a) having a pH of about 7 to about 8; (d) further comprising about 5 mM to about 100 mM of a buffer; (c) further comprising about 0.1 to about 10% w / v cryoprotectant; (d) further comprising about 5 mM to about 150 mM of an inorganic salt; (e) having a payload of about 0.1 mg / mL to about 10 mg / mL.

[0155] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, and (v) a loaded lipid nanoparticle composition comprising a payload comprising a nucleic acid; The loaded lipid nanoparticle composition comprises (a) having a pH of about 7 to about 8; (b) further comprising about 10 mM to about 50 mM of a buffer comprising an acetate buffer and a Tris buffer; (c) further comprising about 1% w / v to about 5% w / v sucrose; (d) further comprising about 50 mM to about 90 mM NaCl; (e) having a payload of about 0.1 mg / mL to about 10 mg / mL.

[0156] Some embodiments comprise the following components: (i) ionized lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, and (v) a loaded lipid nanoparticle composition comprising a payload comprising RNA; The loaded lipid nanoparticle composition comprises (a) having a pH of about 7 to about 8; (b) further comprising about 10 mM to about 50 mM of a buffer comprising an acetate buffer and a Tris buffer; (c) further comprising about 1% to about 5% sucrose; (d) further comprising about 70 mM NaCl; (e) having a payload of about 1 mg / mL to about 2 mg / mL.

[0157] In some embodiments, the empty or filled lipid nanoparticle composition comprises about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, or about 40 mol% to about 50 mol% ionized lipid relative to total lipid.

[0158] In some embodiments, the empty or filled lipid nanoparticle composition comprises about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10 mol% to about 12 mol% phospholipid relative to total lipid.

[0159] In some embodiments, the empty or filled lipid nanoparticle composition comprises about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 37 mol% to about 42 mol% structured lipid relative to total lipid.

[0160] In some embodiments, the empty or filled lipid nanoparticle composition comprises about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1 mol%, or about 0.25 mol% to about 0.75 mol% PEG-lipid relative to the total lipid.

[0161] In some embodiments, the empty or filled lipid nanoparticle composition comprises, relative to total lipid, about 40 mol % to about 50 mol % ionized lipids; about 10 mol% to about 12 mol% phospholipids, about 37 mol % to about 42 mol % structured lipids, and Contains about 0.25 mol% to about 0.75 mol% PEG-lipid.

[0162] In some embodiments, the lipid solution contains, relative to total lipid, Approximately 49 mol% ionized lipids, about 11 mol% to about 12 mol% phospholipids, Approximately 39 mol% structural lipids, and Contains approximately 0.5 mol% PEG-lipid.

[0163] Any empty or filled lipid nanoparticle composition provided herein can be prepared for storage or transportation. For example, the empty or filled lipid nanoparticle composition can be refrigerated, frozen, or lyophilized. In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen, for example, at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C for storage and / or transportation.

[0164] Ionized lipids As used herein, the term "ionized lipid" has its ordinary meaning in the art and may refer to a lipid that contains one or more charged moieties. In some embodiments, an ionized lipid may be positively or negatively charged. For example, an ionized lipid may be positively charged at low pH, in which case it may be referred to as a "cationic lipid". In certain embodiments, an ionized lipid molecule may contain an amine group and may be referred to as an ionized amino lipid. As used herein, a "charged moiety" is a chemical moiety that has a formal electronic charge, for example, monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of a charged moiety may in some cases vary with environmental conditions, e.g., a change in pH may change the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of a molecule may be selected as desired.

[0165] In some embodiments, the nanoparticles described herein comprise about 30 mol% to about 60 mol% ionized lipids. In some embodiments, the nanoparticles described herein comprise about 35 mol% to about 55 mol% ionized lipids. In some embodiments, the nanoparticles comprise about 40 mol% to about 50 mol% ionized lipids. In some embodiments, the nanoparticles comprise about 45 mol% to about 50 mol% ionized lipids.

[0166] In some embodiments, the ionizable lipid is an ionizable amino lipid. In one embodiment, the ionizable amino lipid can have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0167] In some embodiments, the ionizable lipid has formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and [ka] indicates the attachment point, R aα , R aβ , R aγ , and R aδ are independently H, C 2~12 Alkyl, and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from n is selected from 1, 2, 3, 4, and 5; [ka] indicates the attachment point, R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R 6 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1~12 Alkyl or C 2~12 alkenyl, l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0168] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aα , R aβ , R aγ , and R aδare H, respectively, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0169] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aα , R aβ , R aγ , and R aδ are H, respectively, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 3, m is 7.

[0170] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aα is C 2~12 is alkyl, R aβ , R aγ , and R aδ are H, respectively, R 2 and R 3 are C 1~14 is alkyl, R 4 teeth, [ka] and R 10 is -NH(C 1~6 alkyl), n2 is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0171] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aα , R aβ , and R aδ are H, respectively, R aγ is C 2~12 is alkyl, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0172] In some embodiments, the ionizable lipid is [ka] or an N-oxide or salt thereof.

[0173] In some embodiments, the ionizable lipid is a compound: [ka] or an N-oxide or salt thereof.

[0174] In some embodiments, the ionizable lipid is a compound: [ka] or an N-oxide or salt thereof.

[0175] In some embodiments, the ionizable lipid is a compound: [ka] or an N-oxide or salt thereof.

[0176] In some embodiments, the ionizable lipid is a compound: [ka] or an N-oxide or salt thereof.

[0177] In some embodiments, the ionizable lipid has formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and [ka] indicates the attachment point, R aβ , R aγ , and R aδ are independently H, C 2~12 Alkyl, and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R 6 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1~12 Alkyl or C 2~12 alkenyl, l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0178] In some embodiments, the ionizable lipid has formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and [ka] indicates the attachment point, Raα , R aβ , R aγ , and R aδ are independently H, C 2~12 Alkyl, and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; Each R 5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R 6 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1~12 Alkyl or C 2~12 alkenyl, l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0179] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aβ , R aγ , and R aδ are H, respectively, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0180] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aβ , R aγ , and R aδ are H, respectively, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 3, m is 7.

[0181] In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and [ka] indicates the attachment point, R aβ and R aδ are H, respectively, R aγ is C 2~12 is alkyl, R 2 and R 3 are C 1~14 is alkyl, R 4 is -(CH2) n OH, n is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0182] In some embodiments, the ionizable lipid has formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and [ka] indicates the attachment point, R aα , R aβ , R aγ , and R aδ are independently H, C 2~12 Alkyl, and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 teeth, [ka] and [ka] indicates the attachment point, R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; Each R 6 is independently 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1~12 Alkyl or C 2~12 alkenyl, l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0183] In some embodiments, R 1 teeth, [ka] and [ka] indicates the attachment point, R aβ , R aγ , and R aδ are H, respectively, R aα is C 2~12 is alkyl, R 2 and R 3 are C 1~14 is alkyl, R 4 teeth, [ka] and [ka] indicates the attachment point, R 10 is NH(C 1~6 alkyl), n2 is 2, Each R 5 is H, Each R 6 is H, M and M' are each -C(O)O-; R' is C 1~12 is alkyl, l is 5, m is 7.

[0184] In some embodiments, the ionizable lipid of formula (I) is [ka] or an N-oxide or salt thereof.

[0185] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' 環状 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ and R aδ are independently H, C 1~12 Alkyl, and C 2~12 alkenyl, R aγ and R aδ At least one of them is C 1~12 Alkyl and C 2~12 alkenyl, R bγ and R bδ are independently H, C 1~12 Alkyl, and C 2~12 alkenyl, R bγ and R bδ At least one of them is C 1~12 Alkyl and C 2~12 alkenyl, R 2 and R 3 are each independently1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1~12 Alkyl or C 2~12 alkenyl, Y a is C 3~6 is a carbocyclic ring, R * " a is C 1~15 Alkyl and C 2~15 alkenyl, s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0186] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ and R aδ are independently H, C 1~12 Alkyl, and C 2~12 alkenyl, R aγ and R aδ At least one of them is C 1~12 Alkyl and C 2~12 alkenyl, R bγ and R bδ are independently H, C 1~12 Alkyl, and C 2~12 alkenyl, R bγ and R bδ At least one of them is C 1~12 Alkyl and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1~12 Alkyl or C 2~12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0187] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ and R bγ are each independently 1~12 Alkyl and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2)n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1~12 Alkyl or C 2~12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0188] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, Raγ is C 1~12 Alkyl and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R' is C 1~12 Alkyl or C 2~12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0189] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ and R bγ are each independently 1~12 Alkyl and C 2~12 alkenyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2, Each R is independently C 1~6 Alkyl, C 2~3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1~12 Alkyl or C 2~12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0190] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ is C 1~12 Alkyl and C 2~12 alkenyl, R 2 and R 3 are each independently 1~14 Alkyl and C 2~14 alkenyl, R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; R' is C 1~12 Alkyl or C 2~12 alkenyl, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0191] In some embodiments, m and l are each independently selected from 4, 5, and 6. In some embodiments, m and l are each 5.

[0192] In some embodiments, each R' is independently C 1~12 In some embodiments, each R' is independently C 2~5 It is an alkyl.

[0193] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each independently 1~14 It is an alkyl.

[0194] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each independently 6~10 It is an alkyl.

[0195] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each C8 alkyl.

[0196] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 1~12 is alkyl, R 2 and R 3 are each independently 6~10 It is an alkyl.

[0197] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2~6 is alkyl, R 2 and R 3 are each independently 6~10 In some embodiments, R' is alkyl. 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2~6 is alkyl, R 2 and R 3 are each C8 alkyl.

[0198] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ is C 1~12 It is an alkyl.

[0199] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ is C 2~6 It is an alkyl.

[0200] In some embodiments, m and l are each independently selected from 4, 5, and 6; and each R′ is independently C 1~12 In some embodiments, m and l are each 5 and each R' is independently C 2~5 It is an alkyl.

[0201] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently C 1~12 is alkyl, R aγ and R bγ are C 1~12 It is an alkyl.

[0202] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each 5; and each R′ is independently C 2~5 is alkyl, R aγ and R bγ are C 2~6 It is an alkyl.

[0203] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each independently selected from 4, 5 and 6; R′ is C 1~12 is alkyl, R aγ is C 1~12 is alkyl, R 2 and R 3 are each independently 6~10 It is an alkyl.

[0204] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each 5; R′ is C 2~5 is alkyl, R aγ is C 2~6 is alkyl, R 2 and R 3 are each C8 alkyl.

[0205] In some embodiments, R 4 teeth, [ka] and R 10 is NH(C 1~6 alkyl) and n2 is 2.

[0206] In some embodiments, R 4 teeth, [ka] and R 10is NH(CH3) and n2 is 2.

[0207] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1~12 is alkyl, R aγ and R bγ are C 1~12 is alkyl, R 4 teeth, [ka] and R 10 is NH(C 1~6 alkyl) and n2 is 2.

[0208] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each 5; and each R′ is independently 2~5 is alkyl, R aγ and R bγ are C 2~6 is alkyl, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0209] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each independently selected from 4, 5 and 6; R′ is C 1~12 is alkyl, R 2 and R 3 are each independently 6~10 is alkyl, R aγ is C 1~12 is alkyl, R 4 teeth, [ka] and R 10 is NH(C 1~6 alkyl) and n2 is 2.

[0210] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each 5; R′ is C 2~5 is alkyl, R aγ is C 2~6 is alkyl, R 2 and R 3 are each C8 alkyl, and R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0211] In some embodiments, R 4 is -(CH2) n OH and n is 2, 3, or 4. In some embodiments, R 4 is -(CH2) n OH and n is 2.

[0212] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1~12 is alkyl, R aγ and R bγ are C 1~12 is alkyl, R 4 is -(CH2) n OH and n is 2, 3 or 4.

[0213] In some embodiments, R' 分岐鎖 teeth, [ka] and R' b teeth, [ka] m and l are each 5; and each R′ is independently 2~5 is alkyl, R aγ and R bγ are C 2~6 is alkyl, R 4 is -(CH2) n OH and n is 2.

[0214] In some embodiments, the ionizable lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐鎖 or R' 環状 and R' 分岐鎖 teeth [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ is C 1~12 is alkyl, R 2 and R 3 are each independently 1~14 is alkyl, R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; R' is C 1~12 is alkyl, m is selected from 4, 5, and 6; l is selected from 4, 5, and 6.

[0215] In some embodiments, m and l are each 5 and n is 2, 3, or 4.

[0216] In some embodiments, R' is C 2~5 is alkyl, R aγ is C 2~6 is alkyl, R 2 and R 3 are C 6~10 It is an alkyl.

[0217] In some embodiments, m and l are each 5, n is 2, 3, or 4, and R′ is C 2~5 is alkyl, R aγ is C 2~6 is alkyl, R 2 and R 3 are C 6~10 It is an alkyl.

[0218] In some embodiments, the ionizable lipid has formula (II-g): [ka] or an N-oxide or salt thereof, wherein R aγ is C 2~6 is alkyl, R' is C 2~5 is alkyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 3, 4, and 5; R 10 is NH(C 1~6 alkyl), n2 is selected from 1, 2, and 3.

[0219] In some embodiments, the ionizable lipid has formula (II-h): [ka] or an N-oxide or salt thereof, wherein R aγ and R bγ are each independently 2~6 is alkyl, Each R' is independently C 2~5 is alkyl, R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the attachment point, n is selected from 3, 4, and 5; R 10 is NH(C 1~6 alkyl), n2 is selected from 1, 2, and 3.

[0220] In some embodiments, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0221] In some embodiments, R 4 is -(CH2)2OH.

[0222] In some embodiments, the ionizable lipid has formula (III): [ka] or an N-oxide or salt thereof, wherein R1, R2, R3, R4, and R5 are independently C 5~20 Alkyl, C 5~20 alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X 1 , X 2 and X 3 are each independently selected from a bond, -CH-, -(CH)-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH-, -CH-C(O)-, -C(O)O-CH-, -OC(O)-CH-, -CH-C(O)O-, -CH-OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3~6 is a carbocyclic ring, Each R * is independently 1~12 Alkyl and C 2~12 alkenyl, Each R is independently C 1~3 Alkyl and C 3~6 is selected from carbocycles, Each R' is independently C 1~12 Alkyl, C 2~12 alkenyl, and H; Each R" is independently 3~12 Alkyl and C 3~12 alkenyl, i)X 1 , X 2 , and X 3 at least one of is not -CH2-; and / or ii) At least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0223] In some embodiments, R1, R2, R3, R4, and R5 are each C 5~20 is alkyl, and X 1 is -CH2- and X 2 and X 3 are each -C(O)-.

[0224] In some embodiments, the compound of formula (III) is: [ka]

[0225] In some embodiments, the compound of formula (I) is [ka] It is.

[0226] In some embodiments, the ionizable lipid is [ka] [ka] It is.

[0227] mRNA-lipid adducts It has been found that certain ionized lipids are prone to form lipid-polynucleotide adducts. In particular, ionized lipids that contain tertiary amine groups can decompose into one or both of secondary amines and reactive aldehyde species that can interact with polynucleotides (such as mRNA), forming ionized lipid-polynucleotide adduct impurities that can be detected by reversed-phase ion pair chromatography (RP-IP HPLC). For example, oxidation of tertiary amines can form N-oxides, which can undergo acid / base catalyzed hydrolysis at the amine to generate aldehydes and secondary amines that can also form adduct pairs with mRNA. Thus, in some embodiments, the ionized lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity.

[0228] It has also been found that such adducts can interfere with mRNA translation and affect the activity of lipid nanoparticle (LNP) formulated mRNA products.Therefore, it is advantageous to prepare and use LNP compositions with reduced content of ionized lipid-polynucleotide adduct impurities, such as less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the mRNA is in the form of ionized lipid-polynucleotide adduct impurities, as can be measured by RP-IP HPLC.Therefore, according to some embodiments, LNP compositions are provided in which less than about 10%, less than about 5%, or less than about 1% of the mRNA is in the form of ionized lipid-polynucleotide adduct impurities, including less than 10%, less than 5%, or less than 1%, as can be measured by RP-IP HPLC.

[0229] In some embodiments, the amount of lipid aldehyde in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of N-oxide compound in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of transition metal such as Fe in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of halogenated alkyl compound in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of anhydrous compound in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of ketone compound in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of conjugated diene compound in the composition is less than about 50 ppm, including less than 50 ppm.

[0230] In some embodiments, the composition is stable against the formation of ionized lipid-polynucleotide adduct impurities. In some embodiments, the amount of ionized lipid-polynucleotide adduct impurities in the composition increases at an average rate of less than about 2% per day, including an average rate of less than 2% per day, when stored at a temperature of about 25° C. or less. In some embodiments, the amount of ionized lipid-polynucleotide adduct impurities in the composition increases at an average rate of less than about 0.5% per day, including an average rate of less than 0.5% per day, when stored at a temperature of about 5° C. or less. In some embodiments, the amount of ionized lipid-polynucleotide adduct impurities in the composition increases at an average rate of less than about 0.5% per day, when stored at a refrigerated temperature, which is optionally about 5° C.

[0231] Lipid vehicle (e.g., LNP) compositions having reduced content of ionized lipid-polynucleotide adduct impurities can be prepared by methods that inhibit the formation of either or both of N-oxides and aldehydes. Such methods may include treating a composition comprising an ionized lipid that comprises a tertiary amine group to inhibit the formation of either or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent, treating the composition with a chelating agent, adjusting the pH of the composition, adjusting the temperature of the composition, and adjusting a buffer in the composition. Such methods may include one or more of treating the ionized lipid with a scavenger, treating the ionized lipid with a reducing agent, treating the ionized lipid with a chelating agent, treating the polynucleotide with a reducing agent, and treating the polynucleotide with a chelating agent, prior to combining the ionized lipid with the polynucleotide.

[0232] According to any of the above, the scavenger, reducing agent and / or reducing agent may be an agent that reacts with an aldehyde, a ketone, an anhydride and / or a diene compound. The scavenger may include one or more selected from (O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4-[2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), triethylamine (TEA), piperidine 4-carboxylate (BPPC) and combinations thereof. The reducing agent may include a boron compound (e.g., sodium borohydride and / or bis(pinacolato)diboron). The reducing agent may include a boron compound such as one or both of sodium borohydride and bis(pinacolato)diboron. The chelating agent may include immobilized iminodiacetic acid. The reducing agent may include an immobilized reducing agent such as diphenylphosphine immobilized on silica (Si-DPP), thiol immobilized on agarose (Ag-thiol), cysteine ​​immobilized on silica (Si-cysteine), thiol immobilized on silica (Si-thiol), or combinations thereof. The reducing agent may include a free reducing agent such as potassium metabisulfite, sodium thioglycolate, tris(2-carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetylcysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or combinations thereof.

[0233] According to any of the above, the pH may be at or adjusted to a pH of about 7 to about 9.

[0234] According to any of the above, the buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate and TRIS (tris(hydroxymethyl)aminomethane). According to any of the above, the buffer may be TRIS, may be about 20 mM to about 150 mM TRIS, or may be adjusted to about 20 mM to about 150 mM TRIS.

[0235] According to any of the above, the temperature of the composition may be below 25°C or may be adjusted to be below 25°C.

[0236] The composition may also include a free reducing agent or antioxidant.

[0237] PEGylated lipids The PEG lipid component of the lipid nanoparticle composition may comprise one or more molecules that contain polyethylene glycol, e.g., PEG or PEG-modified lipid. Such species may alternatively be referred to as PEGylated lipid. PEG lipid is a lipid that is modified with polyethylene glycol.

[0238] In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.1 mol% to about 10 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.1 mol% to about 5 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.1 mol% to about 3 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.1 mol% to about 2 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.1 mol% to about 1 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.25 mol% to about 0.75 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.5 mol% PEG-lipid.

[0239] The PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be a PEG-c-DOMG, PEG-DMG (e.g., PEG-DMG2000 or DMG-PEG2000), PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0240] In some embodiments, the PEG lipid is PEG-DMG (DMG-PEG or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol). In some embodiments, the PEG lipid is PEG-DMG2000 (or DMG-PEG2000), where 2000 represents the average molecular weight. A representative PEG-DMG structure is: [ka]

[0241] In one embodiment, the PEG lipid may be a PEGylated lipid, such as those described in International Publication No. WO2012 / 099755, the entire contents of which are incorporated herein by reference. Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. A "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid"), as generally defined herein, is a PEGylated lipid that has one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment.

[0242] In certain embodiments, the PEG lipid is a compound of formula (VII): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is an optionally substituted C 1~10 alkylene, wherein the optionally substituted C 1~10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O- or -NR N C(O)N(R N )- and replaced by D is a moiety obtained by click chemistry or cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6One methylene unit of the alkylene is optionally -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O- or -NR N C(O)N(R N )- and replaced by R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl, optionally where R 2 one or more methylene units in are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, Each R N are independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2.

[0243] In certain embodiments, the compound of formula (VII) is a PEG-OH lipid (i.e., R 3 -OR O and R O is hydrogen). In certain embodiments, the compound of formula (VII) has the formula (VII-OH): [ka] or a salt thereof.

[0244] In certain embodiments, D is a moiety obtained by click chemistry (e.g., a triazole). In certain embodiments, the compound of formula (VII) has formula (VII-a-1) or (VII-a-2): [ka] or a salt thereof.

[0245] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0246] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0247] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0248] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0249] In certain embodiments, D is a moiety that is cleavable under physiological conditions (e.g., an ester, an amide, a carbonate, a carbamate, a urea). In certain embodiments, the compound of formula (VII) has formula (VII-b-1) or (VII-b-2): [ka] or a salt thereof.

[0250] In certain embodiments, the compound of formula (VII) has the formula (VII-b-1-OH) or (VII-b-2-OH): [ka] or a salt thereof.

[0251] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0252] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0253] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0254] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0255] In certain embodiments, the PEG lipid is a PEGylated fatty acid. In certain embodiments, the PEG lipid is a compound of formula (VIII). As used herein, formula (VIII): [ka] or a salt thereof, wherein: R 3 -OR O and R O is hydrogen, an optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is an optionally substituted C 10~40 Alkyl, optionally substituted C 10~40 alkenyl, or optionally substituted C 10~40 alkynyl, and optionally R 5 One or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N)S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, R N Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0256] In certain embodiments, the compound of formula (VIII) has the formula (VIII-OH): [ka] or a salt thereof.

[0257] In certain embodiments, the compound of formula (VIII) has the following formula: [ka] or a salt thereof. In some embodiments, r is 43, 44, 45, or 46. In some embodiments, r is 45.

[0258] In yet another embodiment, the compound of formula (VIII) is [ka] or a salt thereof.

[0259] In some embodiments, the compound of formula (VIII) is [ka] It is.

[0260] In certain embodiments, the PEG lipid has the following formula: [ka] In some embodiments, r is 45, or a salt thereof.

[0261] Additional suitable PEG lipids are described in WO2017 / 099823, which is incorporated by reference in its entirety.

[0262] Phospholipids Phospholipids as defined herein are lipids that contain a phosphate group. The lipid component of the lipid nanoparticle composition may comprise one or more phospholipids, for example, one or more (poly)unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. In general, the phospholipids may comprise a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species, including natural species, with modifications and substitutions, including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cell recognition, or for conjugating nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes).

[0263] In some embodiments, the lipid nanoparticle compositions described herein may comprise about 1 mol% to about 20 mol% phospholipids. In some embodiments, the lipid nanoparticle compositions described herein may comprise about 5 mol% to about 15 mol% phospholipids. In some embodiments, the lipid nanoparticle compositions comprise about 8 mol% to about 13 mol% phospholipids. In some embodiments, the lipid nanoparticle compositions comprise about 10 mol% to about 12 mol% phospholipids.

[0264] Suitable phospholipids include the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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-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 (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 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), and sphingomyelin. Each possibility represents a separate embodiment.

[0265] In some embodiments, the phospholipid is DSPC. In certain embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid comprises both DSPC and DOPE. In some embodiments, the phospholipid is 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE) [ka] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC) [ka] 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(sodium salt) (4ME 16:0 PG), or [ka] 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS) [ka] or a mixture thereof.

[0266] Further examples of suitable phospholipids include, but are not limited to, the following: [ka] [ka]

[0267] In certain embodiments, the phospholipid has formula (IX): [ka] or a salt thereof, wherein Each R 1 are independently H or optionally substituted alkyl, or optionally, two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6 One methylene unit of the alkylene is optionally -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O- or -NR N C(O)N(R N )- and replaced by R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl, optionally where R 2 one or more methylene units in are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NRN )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2 (However, this compound has the formula: [ka] It is not something like In the formula, each R 2 are independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0268] In certain embodiments, the phospholipid is an analog or variant of DSPC, such as, for example, a phospholipid having formula (IX): [ka] or a salt thereof, wherein Each R 1 are independently optionally substituted alkyl, or optionally, two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6 One methylene unit of the alkylene is optionally -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O- or -NR N C(O)N(R N )- and replaced by R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl, optionally where R 2one or more methylene units in are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-, R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2. provided that the compound has the formula: [ka] It is not something like In the formula, each R 2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0269] In some embodiments, the compound has the formula: [ka] It is not something like In the formula, each R 2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0270] In certain embodiments, suitable phospholipids include a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, the phospholipid with a modified head group is DSPC, or an analog thereof, with a modified quaternary amine. For example, in the embodiment of formula (IX), R 1 At least one of R 1 In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof, wherein each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each v is independently 1, 2, or 3. In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof.

[0271] In certain embodiments, the compound of formula (IX) has the following formula: [ka] [ka] or a salt thereof.

[0272] In certain embodiments, the compound of formula (IX) has the formula (IX-a): [ka] or a salt thereof.

[0273] In certain embodiments, suitable phospholipids include modified cores. In certain embodiments, the phospholipids having modified cores described herein are DSPCs or their analogs having modified core structures. For example, in certain embodiments of formula (IX-a), group A is represented by the following formula: [ka] It is not one of.

[0274] In certain embodiments, the compound of formula (IX-b-4) has the following formula: [ka] or a salt thereof.

[0275] In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof.

[0276] In certain embodiments, the phospholipid comprises a cyclic moiety instead of a glyceride moiety. In certain embodiments, the phospholipid is DSPC, or an analog thereof, having a cyclic moiety instead of a glyceride moiety. In certain embodiments, the compound of formula (IX) has formula (IX-b): [ka] or a salt thereof.

[0277] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-1): [ka] or a salt thereof, wherein: w is 0, 1, 2, or 3.

[0278] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-2): [ka] or a salt thereof.

[0279] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-3): [ka] or a salt thereof.

[0280] In certain embodiments, the compound of formula (Ib) has the formula (Ib-4): [ka] or a salt thereof.

[0281] In certain embodiments, the compound of formula (IX-b) has the following formula: [ka] or a salt thereof.

[0282] In certain embodiments, suitable phospholipids include modified tails. In certain embodiments, the phospholipid is DSPC or an analog thereof with modified tail. As described herein, a "modified tail" may be a tail with a shorter or longer aliphatic chain, a branched aliphatic chain, a substituted aliphatic chain, an aliphatic chain with one or more methylenes replaced with a cyclic or heteroatom group, or any combination thereof. For example, in certain embodiments, the compound of (IX) is of formula (IX-a) or a salt thereof, wherein R 2 In at least one case, R 2 Each instance of is optionally replaced by C 1~30 alkyl, where R 2 one or more methylene units in are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(RN )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by

[0283] In certain embodiments, the compound of formula (IX) has the formula (IX-c): [ka] or a salt thereof, wherein: each x is independently an integer between 0 and 30, inclusive; In each instance, G is an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NRN C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-. Each possibility represents a separate embodiment.

[0284] In certain embodiments, the compound of formula (IX-c) has the formula (IX-c-1): [ka] or a salt thereof, wherein Each instance of v is independently 1, 2, or 3.

[0285] In certain embodiments, the compound of formula (IX-c) has the formula (IX-c-2): [ka] or a salt thereof.

[0286] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0287] In certain embodiments, the compound of formula (IX-c) is: [ka] or a salt thereof.

[0288] In certain embodiments, the compound of formula (IX-c) has the formula (Ic-3): [ka] or a salt thereof.

[0289] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0290] In certain embodiments, the compound of formula (IX-c) is: [ka] or a salt thereof.

[0291] In certain embodiments, a suitable phospholipid comprises a modified phosphocholine moiety, where the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, the phospholipid is a compound of formula (IX), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof.

[0292] In certain embodiments, the compound of formula (IX) has the following formula: [ka] [ka] or a salt thereof.

[0293] In certain embodiments, alternative lipids are used in place of phospholipids. Non-limiting examples of such alternative lipids include the following: [ka] Examples include:

[0294] structural lipids The lipid nanoparticle composition may include one or more structured lipids. Incorporating a structured lipid into a lipid nanoparticle may help reduce aggregation of other lipids within the particle. The structured lipid may be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is an analog of cholesterol. In certain embodiments, the structured lipid is alpha-tocopherol. Examples of structured lipids include, but are not limited to, the following: [ka]

[0295] In some embodiments, the lipid nanoparticle compositions described herein may comprise about 20 mol% to about 60 mol% structured lipid. In some embodiments, the lipid nanoparticle compositions comprise about 30 mol% to about 50 mol% structured lipid. In some embodiments, the lipid nanoparticle compositions comprise about 35 mol% to about 45 mol% structured lipid. In some embodiments, the lipid nanoparticle compositions comprise about 37 mol% to about 42 mol% structured lipid. In some embodiments, the lipid nanoparticle compositions comprise about 35, about 36, about 37, about 38, about 39, or about 40 mol% structured lipid. In some embodiments, the nanoparticles comprise about 39 to about 40 mol% structured lipid. In some embodiments, the structured lipid is cholesterol or the following structure: [ka] It is a compound having the formula:

[0296] Therapeutic and prophylactic agents as payloads The lipid nanoparticle composition of the present disclosure can be used to deliver a wide variety of different therapeutic or prophylactic agents to patients. The therapeutic agents delivered by the composition are nucleic acids, but non-nucleic acid agents such as small molecules, chemotherapeutic drugs, peptides, polypeptides, and other biomolecules are also payloads encompassed by the present disclosure. Nucleic acids that can be delivered include DNA-based molecules (i.e., containing deoxyribonucleotides) and RNA-based molecules (i.e., containing ribonucleotides). Furthermore, the nucleic acid can be the naturally occurring form of the molecule or a chemically modified form of the molecule (e.g., containing one or more modified nucleotides).

[0297] In one embodiment, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors).

[0298] In one embodiment, the therapeutic agent is a DNA therapeutic agent.The DNA molecule can be a double-stranded DNA, a single-stranded DNA (ssDNA), or a molecule that is partially double-stranded DNA, i.e., has a part that is double-stranded and a part that is single-stranded.In some cases, the DNA molecule is triple-stranded or partially triple-stranded, i.e., has a part that is triple-stranded and a part that is double-stranded.The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

[0299] A DNA therapeutic agent can be a DNA molecule capable of introducing a gene into a cell, for example, a DNA molecule capable of encoding a transcript and expressing it. In some embodiments, the DNA molecule can be naturally derived, for example, isolated from a natural source. In other embodiments, the DNA molecule is a synthetic molecule, for example, a synthetic DNA molecule generated in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0300] The DNA therapeutic agent, e.g., DNA vector, described herein may include a variety of different features. The DNA therapeutic agent, e.g., DNA vector, described herein may include a non-coding DNA sequence. For example, the DNA sequence may include at least one regulatory element of a gene, e.g., a promoter, an enhancer, a termination element, a polyadenylation signal element, a splicing signal element, etc. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, the DNA sequence described herein may have a non-coding DNA sequence operably linked to a transcriptionally active gene. In other embodiments, the DNA sequence described herein may have a non-coding DNA sequence that is not linked to a gene. That is, the non-coding DNA does not control a gene on the DNA sequence.

[0301] In one embodiment, the therapeutic agent is an RNA therapeutic agent.The RNA molecule can be single-stranded RNA, double-stranded RNA (dsRNA), or a molecule that is partially double-stranded RNA, i.e., has a part that is double-stranded and a part that is single-stranded.The RNA molecule can be a circular RNA molecule or a linear RNA molecule.

[0302] The RNA therapeutic agent can be an RNA therapeutic agent that can introduce a gene into cells, for example, that can code a protein of interest, thereby increasing the expression of the protein of interest in airway cells.In some embodiments, the RNA molecule can be naturally derived, for example, isolated from a natural source.In other embodiments, the RNA molecule is a synthetic molecule, for example, a synthetic RNA molecule that is produced in vitro.

[0303] Non-limiting examples of RNA therapeutics include messenger RNA (mRNA) (e.g., encoding a protein of interest), modified mRNA (mmRNA), mRNA with incorporated microRNA binding site(s) (miR binding site(s)), modified RNA containing functional RNA elements, microRNA (miRNA), antagomir, small interfering RNA (siRNA) (including shortmers and Dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology, each of which is further described in the subsections below.

[0304] mRNA may be naturally occurring or non-naturally occurring. mRNA may contain one or more modified nucleic acid bases, nucleosides, or nucleotides, as shown below, in which case it may be referred to as "modified mRNA" or "mmRNA". As described herein, "nucleoside" is defined as a compound that includes a sugar molecule (e.g., pentose or ribose) or its derivative in combination with an organic base (e.g., purine or pyrimidine) or its derivative (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside that includes a phosphate group.

[0305] An mRNA may include a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleic acid bases, nucleosides, or nucleotides may be analogs of the standard species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified.

[0306] In some embodiments, the mRNAs described herein may include a 5' cap structure, chain terminating nucleotides, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0307] The 5' cap structure or cap species is a compound that includes two nucleoside moieties linked by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). The cap species may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide linked at the 5' position by a triphosphate bond and methylated at the 7th position, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. The cap species may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, and m27,O2'GppppG.

[0308] Alternatively or additionally, the mRNA may include chain-terminating nucleosides. For example, chain-terminating nucleosides may include nucleosides deoxygenated at the 2' and / or 3' positions of the sugar group. Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymine, and 2',3' dideoxynucleosides, such as 2',3' dideoxyadenosine, 2',3' dideoxyuridine, 2',3' dideoxycytosine, 2',3' dideoxyguanosine, and 2',3' dideoxythymine. In some embodiments, the introduction of chain-terminating nucleotides into the mRNA, for example at the 3' end, may result in stabilization of the mRNA, for example as described in International Patent Publication No. WO2013 / 103659.

[0309] The mRNA may alternatively or additionally comprise a stem loop, such as a histone stem loop. The stem loop may comprise 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, the stem loop may comprise 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or in, before, or after a polyA sequence or tail. In some embodiments, the stem loop may affect one or more functions (or functions) of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0310] The mRNA may alternatively or additionally comprise a polyA sequence and / or a polyadenylation signal. The polyA sequence may be completely or mainly composed of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the mRNA. In some embodiments, the polyA sequence may affect the nuclear transport, translation, and / or stability of the mRNA.

[0311] The mRNA may alternatively or additionally contain a microRNA binding site.

[0312] In some embodiments, the mRNA is a bicistronic mRNA that includes a first coding region and a second coding region (with an intervening sequence that includes an internal ribosome entry site (IRES) sequence that allows internal translation initiation between the first and second coding regions, or an intervening sequence that encodes a self-cleaving peptide, such as a 2A peptide). IRES sequences and 2A peptides are commonly used to increase the expression of multiple proteins from the same vector. A variety of IRES sequences are known and available in the art and can be used, including, for example, the encephalomyocarditis virus IRES.

[0313] In some embodiments, the mRNA of the present disclosure comprises one or more modified nucleic acid bases, nucleosides, or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, the modified mRNA may have useful properties compared to a reference unmodified mRNA, including improved stability, intracellular retention, enhanced translation, and / or substantially no induction of the innate immune response of a cell into which the mRNA is introduced. Thus, the use of modified mRNA may improve the efficiency of protein production, intracellular retention of nucleic acids, as well as reduced immunogenicity.

[0314] In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may be reduced in degradation in a cell into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0315] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-Methoxy-uridine (mo5U), Uridine 5-oxyacetic acid (cmo5U), Uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2- Thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (ncm5Um), uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0316] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydro-cytidine (5-hydro-cytidine), 5-amino-cytidine (5-amino ... hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl- These include tyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0317] In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having modified adenines include a-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, Aminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonine (threonine) Onylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0318] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include a-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OhyW), undermodified hydroxywyosine (OhyW), and undermodified hydroxywyosine (OhyW). *), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guano N-methyl-guanosine (m7G), 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2 ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), These include 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0319] In some embodiments, an mRNA of the disclosure comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0320] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases). In one embodiment, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the mRNA of the present disclosure is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) represents 75-100% of the uracils in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) represents 100% of the uracils in the mRNA.

[0321] In some embodiments, the modified nucleobase is modified cytosine.Exemplary nucleobases and nucleosides with modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.In some embodiments, the mRNA of the present disclosure comprises one or more combinations of the above modified nucleobases (e.g., a combination of two, three, or four of the above modified nucleobases).

[0322] In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides with modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A). In some embodiments, the mRNA of the present disclosure comprises one or more combinations of the above modified nucleobases (e.g., combinations of two, three, or four of the above modified nucleobases).

[0323] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. In some embodiments, the mRNA of the present disclosure comprises one or more combinations of the modified nucleobases described above (e.g., a combination of two, three, or four of the modified nucleobases described above).

[0324] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the mRNA of the disclosure comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0325] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0326] In certain embodiments, the mRNA of the present disclosure is uniformly modified for a particular modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), which means that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, the mRNA of the present disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with modified residues such as those described above.

[0327] In some embodiments, the mRNA of the present disclosure may be modified in the coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, the mRNA may be modified in a region other than the coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR are provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0328] Examples of nucleoside modifications and combinations thereof that may be present in the mRNAs of the present disclosure include, but are not limited to, those described in PCT Patent Application Publications WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0329] The mRNA of the disclosure may contain a combination of modifications to the sugar, nucleobase, and / or internucleoside linkages, which may include any one or more of the modifications described herein.

[0330] When a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of that A, U, G, or C nucleotide or nucleoside that is modified. When percentages are listed, they represent the percentage of that particular A, U, G, or C nucleobase triphosphate relative to the total amount of A, U, G, or C triphosphates present. For example, the combination: 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine triphosphates are 5-aminoallyl-CTP and 75% of the cytosines are CTP, and 25% of the uracils are 5-methoxyUTP and 75% of the uracils are UTP. When no modified UTP is listed, naturally occurring ATP, UTP, GTP, and / or CTP are used at 100% of the sites of such nucleotides found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unmodified.

[0331] The mRNA or regions thereof of the present disclosure may be codon-optimized. Methods of codon optimization are known in the art and may be useful for a variety of purposes: to match codon frequencies in target and host organisms and ensure proper folding, to bias GC content to increase mRNA stability or reduce secondary structures, to minimize runs of tandem repeat codons or bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein trafficking sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein, to add, remove, or swap protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust the translation rate and correctly fold various domains of the protein, or to reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, Calif.), and / or proprietary methods. In one embodiment, the mRNA sequence is optimized using an optimization algorithm, for example, to optimize expression in mammalian cells or to improve mRNA stability.

[0332] In certain embodiments, the disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.

[0333] The mRNA of the present disclosure can be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzyme (IVT), solid phase, liquid phase, complex synthesis, small area synthesis, and ligation methods can be utilized. In one embodiment, the mRNA is produced using IVT enzymatic synthesis. Methods for producing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety. Thus, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to in vitro transcribe the mRNA described herein.

[0334] Non-natural modified nucleobases may be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications may be at the internucleoside linkage, the purine or pyrimidine base, or the sugar. In certain embodiments, the modifications may be introduced at the end of the polynucleotide chain or elsewhere in the polynucleotide chain by chemical synthesis or by a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0335] Either enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof with various functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0336] In some embodiments, the payload therapeutic agent is a therapeutic agent that reduces (i.e., decreases, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to reduce protein expression include mRNA with microRNA binding site(s) (miR binding site), microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology.

[0337] In some embodiments, the therapeutic agent is a peptide therapeutic agent. In one embodiment, the therapeutic agent is a polypeptide therapeutic agent.

[0338] In some embodiments, the peptide or polypeptide is naturally occurring, e.g., isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g., a synthetic peptide or polypeptide generated in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In one embodiment, the peptide or polypeptide therapeutic of the composition is a naturally occurring peptide or polypeptide. In one embodiment, the peptide or polypeptide therapeutic of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g., contains fewer than 3, fewer than 5, fewer than 10, fewer than 15, fewer than 20, or fewer than 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0339] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising any of the lipid nanoparticle compositions described herein together with one or more pharma- ceutically acceptable excipients.

[0340] The pharmaceutical composition may optionally contain one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. The pharmaceutical composition of the present disclosure may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents are discussed, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety. In some embodiments, the compositions are administered to a human, human patient, or human subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to a nanoparticle that includes a polynucleotide or polypeptide payload that is delivered as described herein.

[0341] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the steps of bringing the nanoparticles into association with an excipient and / or one or more other accessory ingredients, and then dividing, shaping, and / or packaging the product into the desired single or multiple dose units, as necessary and / or desired.

[0342] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to a subject and / or an appropriate fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0343] The relative amounts of the active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0344] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals.

[0345] As used herein, pharma- ceutically acceptable additives include, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersing or suspending aids, diluents, granulating and / or dispersing agents, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, binders, lubricants or oils, colorants, sweeteners or flavoring agents, stabilizers, antioxidants, antibacterial or antifungal agents, osmolality adjusters, pH adjusters, buffers, chelating agents, cryoprotectants, and / or bulking agents, suitable for the particular dosage form desired. Various additives for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, incorporated herein by reference in its entirety).

[0346] Oxidation is a potential degradation pathway for mRNA, especially liquid mRNA formulations. To prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, and the like, and combinations thereof.

[0347] The pharmaceutical composition can be administered in an effective amount to produce a desired biological effect, e.g., a therapeutic or prophylactic effect (e.g., by expression of a normal gene product to replenish or replace a defective protein or to reduce undesirable protein expression, in some embodiments measured by the alleviation of one or more symptoms). The formulation may be administered in an effective amount to deliver the LNP.

[0348] Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and methods of administration. In some embodiments, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0349] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage forms contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, oral compositions may contain additional therapeutic and / or prophylactic agents, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or aromatic agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0350] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0351] The injectable preparations may be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0352] The pharmaceutical composition may be prepared, packaged, and / or sold as a formulation suitable for pulmonary administration. Such a formulation may include dry particles containing the active ingredient. Such a composition may be in the form of a dry powder for administration using a device including a dry powder reservoir to which a stream of propellant can be directed to disperse the powder, and / or using a self-propelling solvent / powder dispensing container, such as a device including the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions may include a solid fine powder diluent, such as sugar, and may be provided in a unit dose form.

[0353] Low boiling propellants generally include a liquid propellant having a boiling point at atmospheric pressure of about 65° F. or less. The propellant may comprise 50% to 99.9% (w / w) of the composition, and the active ingredient may comprise 0.1% to 20% (w / w) of the composition. The propellant may further include additional ingredients such as liquid non-ionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient).

[0354] Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as an optionally sterile, aqueous and / or dilute alcoholic solution and / or suspension containing the active ingredient, and may be conveniently administered using any nebulizer and / or atomizer device. Such formulations may further comprise one or more additional components, including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate. The droplets provided by this route of administration may have an average diameter ranging from about 1 nm to about 200 nm.

[0355] The formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of pharmaceutical compositions.Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm.Such a formulation is administered by rapid inhalation through the nasal cavity from a container of powder held close to the nose.Formulations suitable for nasal administration may, for example, comprise as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may comprise one or more additional ingredients described herein.

[0356] Methods for Treating or Preventing Disease The loaded lipid nanoparticles described herein may be useful for treating or preventing diseases. In particular, such compositions may be useful in treating diseases characterized by missing or abnormal protein or polypeptide activity. In some embodiments, loaded lipid nanoparticle compositions described herein loaded with mRNA encoding a missing or abnormal polypeptide may be administered or delivered to cells. Subsequent translation of the mRNA produces a polypeptide, thereby reducing or eliminating problems caused by the lack of activity or abnormal activity caused by the polypeptide. Because translation can occur rapidly, the methods and compositions may also be useful for treating acute diseases, disorders, or conditions, such as sepsis, stroke, and myocardial infarction. The therapeutic and / or prophylactic agents contained in the LNPs may also be able to alter the transcription rate of a given species, thereby affecting gene expression.

[0357] Diseases characterized by dysfunctional or abnormal protein or polypeptide activity for which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiac and renal vascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions may be characterized by a lack of protein activity (or a substantial reduction such that proper protein function does not occur). Such proteins may be absent or essentially non-functional. The present disclosure provides a method for treating such diseases, disorders, and / or conditions in a subject by administering LNPs comprising RNA and lipid components including lipids according to formula (I), phospholipids (optionally unsaturated), PEG lipids, and structured lipids, where the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the abnormal protein activity present in the subject's cells.

[0358] The present disclosure provides methods that include administering lipid nanoparticle compositions loaded with one or more therapeutic and / or prophylactic agents, such as nucleic acids and pharmaceutical compositions comprising the same. The terms therapeutic and prophylactic agents can be used interchangeably herein for features and embodiments of the present disclosure. The therapeutic composition, or imaging, diagnostic, or prophylactic composition thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition, and / or for any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of administration; the particular composition; the mode of administration, and the like. The compositions according to the present disclosure may be formulated into unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective, prophylactically effective, or otherwise (e.g., for imaging) appropriate dosage level for any particular patient will depend on a variety of factors, including the severity and identity (if any) of the disorder being treated; the one or more therapeutic and / or prophylactic agents used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the particular pharmaceutical composition used; the duration of treatment; drugs used in combination or concomitantly with the particular pharmaceutical composition used; and similar factors well known in the medical arts.

[0359] Combination therapy Lipid nanoparticle compositions loaded with one or more payload therapeutic and / or prophylactic agents, such as nucleic acids, can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By "combined with," it is not intended to imply that the agents must be administered simultaneously and / or formulated together for delivery, although these delivery methods are within the scope of the present disclosure. The lipid nanoparticle composition may be administered simultaneously with, prior to, or after one or more other desired therapeutic or medical procedures. Generally, each agent is administered at a dose and / or time schedule determined for that agent. In some embodiments, the present disclosure encompasses delivery of the composition, or its imaging, diagnostic, or prophylactic composition, in combination with agents that improve bioavailability, reduce and / or modulate metabolism, inhibit excretion, and / or modulate distribution in the body.

[0360] It will be further understood that therapeutically, prophylactically, diagnostically, or imaging-effective agents utilized in combination may be administered together as a single composition, or separately as different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than the levels at which they are utilized individually. The particular combination of therapies (therapeutic agents or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures with the desired therapeutic effect to be achieved. It will also be understood that the therapies utilized can achieve the desired effect for the same disorder (e.g., a composition useful for treating cancer can be administered simultaneously with a chemotherapeutic agent) or can achieve different effects (e.g., control of any adverse effects, such as infusion reactions).

[0361] The loaded lipid nanoparticle composition may be used in combination with an agent that increases the efficacy and / or therapeutic window of the composition. Such an agent may be, for example, an anti-inflammatory compound, a steroid (e.g., a corticosteroid), a statin, an estradiol, a BTK inhibitor, an S1P1 agonist, a glucocorticoid receptor modifier (GRM), or an antihistamine. In some embodiments, the lipid nanoparticle composition may be used in combination with dexamethasone, methotrexate, acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. In some embodiments, the method of treating a subject in need of treatment or the method of delivering a therapeutic and / or prophylactic agent to a subject (e.g., a mammal) may include pretreating the subject with one or more agents prior to administering the lipid nanoparticle composition.

[0362] Kits and Devices The present disclosure provides kits for conveniently and / or effectively using the lipid nanoparticle compositions of the present disclosure. Typically, the kits include sufficient quantities and / or numbers of components to allow a user to perform multiple treatments of a subject(s) and / or to perform multiple experiments.

[0363] In one aspect, the present disclosure provides a kit comprising the nanoparticles of the present disclosure.

[0364] The kit may further include packaging and instructions and / or a delivery agent for forming the formulation composition. The delivery agent may include saline, a buffer solution, or a lipidoid.

[0365] In some aspects, the kit may include an empty lipid nanoparticle composition and a nucleic acid solution. In some aspects, the kit includes a first container that includes the empty lipid nanoparticle composition and a second container that includes a solution that includes a therapeutic or prophylactic agent. In some aspects, the kit further includes instructions for combining (e.g., mixing) the contents of the first container and the second container. In some embodiments, the container may include a polytetrafluoroethylene (PTFE) bag.

[0366] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0367] The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which a plurality, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0368] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more," and "at least one," may be used interchangeably herein. In certain aspects, the terms "a" or "an" mean "single." In other aspects, the terms "a" or "an" include "two or more" or "plural."

[0369] Furthermore, "and / or" as used herein should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" when used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (single), B (single), and C (single).

[0370] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general definition of many of the terms used in this disclosure.

[0371] Units, prefixes, and symbols are expressed in the form recognized by their International System of Units (SI). Numeric ranges are inclusive of the numbers that define the range. When a range of values ​​is listed, it is understood that each intervening integer value, and each fraction thereof, between the upper and lower recited values ​​of the range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded from the range, and each range in which either, neither, or both limits are included is also encompassed within the disclosure. When values ​​are explicitly listed, it is understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the disclosure. When combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the disclosure. Conversely, when different elements or groups of elements are individually disclosed, the combinations are also disclosed. When any element of the disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded alone or in any combination with the other alternatives are also disclosed herein. More than one element of the disclosure may have such an exclusion, and all combinations of elements having such an exclusion are disclosed herein.

[0372] The term "about" when used in connection with numerical values ​​throughout this specification and claims, unless otherwise specified, refers to an interval of accuracy well known and accepted by those of skill in the art, such as, for example, an interval of accuracy of ±10%.

[0373] Where ranges are given, the endpoints are included. Additionally, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can be taken to be up to one tenth of the unit of the lower limit of that range for any particular value or subrange within the stated range in different embodiments of the present disclosure, unless the context clearly indicates otherwise.

[0374] As used herein, the term "administered in combination" or "co-administration" or "combination therapy" means that two or more agents are administered to a subject at the same time or within an interval such that the effects of each agent on the patient can overlap. In some embodiments, they are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of each other. In some embodiments, the administration of the agents is spaced sufficiently close to each other so that a combined (e.g., synergistic) effect is obtained.

[0375] As used herein, the term "compound" is intended to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" refers to any geometric isomer (e.g., cis and trans isomers), enantiomers, or diastereomers of a compound. The present disclosure encompasses any and all stereoisomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds and means for resolving them into their component enantiomers or stereoisomers are well known. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules by conventional methods to produce solvates and hydrates.

[0376] As used herein, the term "delivering" means providing an entity to a destination. For example, delivery of a polynucleotide to a subject may include administering to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes) a nanoparticle composition comprising the polynucleotide. Administration of a nanoparticle composition to a mammal or mammalian cells may include contacting one or more cells with a lipid nanoparticle composition.

[0377] As used herein, "delivery agent" refers to any substance that at least partially facilitates in vivo, in vitro or ex vivo delivery of a polynucleotide to a target cell.

[0378] As used herein, the term "effective amount" of an agent is an amount sufficient to produce a beneficial or desired result, e.g., a clinical result, and thus the "effective amount" depends on the context in which it is applied. For example, in the context of administering an agent to treat a protein deficiency, the effective amount of the agent is the amount of mRNA expressing said protein sufficient to alleviate, reduce, eliminate, or prevent the signs and symptoms associated with the protein deficiency, e.g., compared to the severity of the symptoms observed when the agent is not administered. The term "effective amount" may be used interchangeably with "effective dose", "therapeutically effective amount", or "therapeutically effective dose".

[0379] As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of a nanoparticle composition compared to the initial total amount of polynucleotide used in the synthesis of the nanoparticle composition. For example, if 97 mg of polynucleotide is encapsulated in the nanoparticle composition out of a total amount of 100 mg of polynucleotide initially provided in the composition, the encapsulation efficiency can be 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial entrapment, confinement, surrounding, or inclusion.

[0380] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription), (2) processing of the mRNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the mRNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0381] As used herein, "linker" refers to a group of atoms, e.g., 10-1,000 atoms, and may be composed of atoms or groups, e.g., but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can attach a modified nucleoside or modified nucleotide on the nucleobase or sugar moiety at a first end and a payload, e.g., a detectable substance or therapeutic agent, at a second end. The linker can be of sufficient length so as not to interfere with introduction into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form polynucleotide multimers (e.g., through the attachment of two or more chimeric polynucleotide molecules or IVT polynucleotides) or polynucleotide conjugates, and to administer a payload as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl (each of which can be optionally substituted as described herein). Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, and derivatives thereof. Other examples include, but are not limited to, cleavable moieties in the linker, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved, for example, using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and / or photolysis, and ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0382] As used herein, the term "lipid amine" refers to a lipid molecule having one or more amine functional groups attached thereto. The amine functional groups can include one or more primary (NH2), secondary (NHR), or tertiary amine groups (NR2), where R represents a non-hydrogen group such as an alkyl group, a carbocyclic group, a heterocyclic group, or a substituted derivative thereof. Lipid amines include sterol amines, where the lipid portion of the molecule is a steroid, such as cholesterol or a related moiety.

[0383] As used herein, the phrase "a moiety cleavable under physiological conditions" refers to, for example, an ester, amide, carbonate, carbamate, or urea moiety.

[0384] As used herein, a "patient" refers to a subject (e.g., a human subject) who desires or requires treatment, is in need of treatment, is undergoing treatment, is to receive treatment, or is receiving care from a skilled professional for a particular disease or condition.

[0385] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0386] The phrase "pharmaceutical acceptable additives" as used herein refers to any component other than the compounds described herein (e.g., a vehicle that can suspend or dissolve active compounds), and has the properties of being substantially non-toxic and non-inflammatory in patients.Additives can include, for example, anti-adhesive agents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), softeners, emulsifiers, excipients (diluents), film-forming agents or coating agents, flavors, fragrances, flow agents (glidants), lubricants, preservatives, printing inks, absorbents, suspending agents, dispersing agents, sweeteners, and hydration water.

[0387] The present disclosure also includes salts of the compounds described herein. As used herein, "salt" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. In some embodiments, the salt is a pharmaceutically acceptable salt. A list of pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17, pp. 171-175, 2002. th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0388] The term "polynucleotide," as used herein, refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). The term also includes modified forms of polynucleotides, e.g., by alkylation and / or by capping, as well as unmodified forms. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing normucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in an arrangement that allows for base pairing and base stacking, such as those found in DNA and RNA. In certain embodiments, the polynucleotide includes an mRNA. In another embodiment, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one unnatural nucleobase. In some embodiments, all of a particular type of nucleobase is replaced with a non-natural nucleobase (e.g., all uridines in the polynucleotides disclosed herein can be replaced with a non-natural nucleobase, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or DNA) contains only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of synthetic DNA, or A, C, G, and U (uridine) in the case of synthetic RNA.

[0389] Those skilled in the art will understand that the T base in the codon maps disclosed herein is present in DNA, whereas in the corresponding RNA, the T base is replaced with a U base. For example, the codon-nucleotide sequences disclosed herein in DNA form, such as vectors or in-vitro translation (IVT) templates, have a T base that is transcribed as a U based on its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (including T) and their corresponding mRNA sequences (including U) are considered codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also understand that equivalent codon maps can be created by replacing one or more bases with unnatural bases. Thus, for example, the TTC codon (DNA map) corresponds to the UUC codon (RNA map), which in turn corresponds to the ΨΨC codon (RNA map in which U is replaced with pseudouridine).

[0390] Canonical AT and GC base pairs are formed under conditions that allow hydrogen bond formation between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy, N3 and C4-NH2 of cytidine and the C2-NH2, N'-H and C6-oxy of guanosine, respectively. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications result in the nucleoside base not effectively forming a canonical base pair with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in a modified nucleotide that does not base pair efficiently with guanosine but will base pair with isoguanosine (Collins et al., U.S. Pat. No. 5,681,702). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides can be prepared using the methods described by Switzer et al., 1993 (supra) and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Patent No. 5,780,610 to Collins et al. Other unnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione).Other such modified nucleotide units which form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al. (supra).

[0391] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may contain modified amino acids. The term also includes amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are polypeptides that contain one or more analogs of amino acids, including, for example, homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and unnatural amino acids such as creatine, as well as other modifications known in the art.

[0392] The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides may be monomeric or may be multi-molecular complexes, such as dimers, trimers, or tetramers. Polypeptides may also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" may be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0393] As used herein, the term "prevent" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, traits, or clinical symptoms of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, traits, or symptoms of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition; and / or reducing the risk of developing a pathology associated with an infection, disease, disorder, and / or condition.

[0394] As used herein, "prophylactic" refers to a therapeutic agent or course of action used to prevent the onset, progression, or spread of a disease.

[0395] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, farm animals, livestock, zoo animals, sport animals, pet animals (such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.), primates (such as ape-men, monkeys, orangutans, and chimpanzees), canines (such as dogs and wolves), felines (such as cats, lions, and tigers), equines (such as horses, donkeys, and zebras), bears, food animals (such as cows, pigs, and sheep), ungulates (such as deer and giraffes), rodents (such as mice, rats, hamsters, and guinea pigs), and the like. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In certain embodiments, the subject is a human patient in need of treatment.

[0396] As used herein, the term "substantially" refers to a qualitative state exhibiting all or nearly all extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical features rarely, if ever, are perfect and / or progress to perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical features.

[0397] The term "therapeutic agent" refers to an agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. For example, in some embodiments, an mRNA encoding a polypeptide can be a therapeutic agent.

[0398] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., a nucleic acid, a drug, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate, diagnose, prevent, and / or delay the onset of the signs and symptoms of the infection, disease, disorder, and / or condition.

[0399] As used herein, the term "treating" or "treatment" or "therapy" refers to the partial or complete alleviation, amelioration, mitigation, delay in onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more signs and symptoms or characteristics of a disease, e.g., cystic fibrosis. For example, "treating" cystic fibrosis can refer to reducing signs and symptoms associated with the disease, extending the lifespan of a patient (increasing survival rate), reducing the severity of the disease, preventing or delaying the onset of the disease, etc. Treatment can be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0400] As used herein, the term "alkyl" or "alkyl group" means a straight-chain or branched saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms).

[0401] As used herein, the term "alkylene" refers to a linking alkyl group.

[0402] As used herein, the term "alkenyl" or "alkenyl group" means a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one double bond.

[0403] As used herein, the term "alkynyl" or "alkynyl group" means a straight or branched chain hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one triple bond.

[0404] As used herein, the terms "carbocycle", "carbocyclyl" and "carbocyclic group" are interchangeable and refer to a monocyclic or polycyclic ring system containing one or more rings of carbon atoms. The ring may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-membered. A carbocycle may be aromatic or non-aromatic, or it may contain both aromatic and non-aromatic rings, in which case the ring is polycyclic.

[0405] As used herein, the term "cycloalkyl" refers to non-aromatic carbocycles and represents a subset of carbocycles. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0406] As used herein, the term "carbocyclylene" refers to a linking carbocyclyl group.

[0407] "C 3~6 The term "carbocycle" refers to a carbocycle including a monocyclic ring having 3 to 6 carbon atoms. A carbocycle may contain one or more double bonds and may be aromatic (e.g., an aryl group). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. A carbocycle may be optionally substituted.

[0408] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group. An example carbocyclylalkyl group is benzyl.

[0409] As used herein, the term "heterocycle", "heterocyclyl" or "heterocyclic group" refers to a monocyclic or polycyclic ring system containing one or more rings, at least one of which contains at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The ring may be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12-membered. The heterocycle may contain one or more double bonds and may be aromatic (e.g., a heteroaryl group). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The heterocycle may be optionally substituted.

[0410] As used herein, the term "heterocycloalkyl" refers to non-aromatic heterocycles and represents a subset of heterocycles. Exemplary heterocycloalkyl groups include azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, and the like.

[0411] As used herein, the term "heterocyclylene" refers to a linking heterocyclyl group.

[0412] As used herein, an "aryl group" is a carbocyclic group that contains one or more carbocyclic aromatic rings. Examples of aryl groups include phenyl and naphthyl groups.

[0413] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group.

[0414] As used herein, the term "arylene" refers to a linking aryl group.

[0415] As used herein, a "heteroaryl group" is a heterocyclic group that contains one or more heteroaromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted.

[0416] As used herein, the term "heteroarylene" refers to a linking heteroaryl group.

[0417] As used herein, the term "oxygen protecting group" refers to an oxo substituent that can be selectively removed under certain conditions (e.g., acidic or basic conditions). Exemplary oxygen protecting groups can include optionally substituted alkyl, carbocyclyl, heterocyclyl, carbocyclylalkyl, and heterocyclylalkyl groups.

[0418] As used herein, the term "nitrogen protecting group" refers to a nitrogen substituent (e.g., an amino substituent) that can be selectively removed under certain conditions (e.g., acidic or basic conditions). In some embodiments, the nitrogen protecting group is 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc).

[0419] Alkyl, alkenyl, alkynyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. Optional substituents include halogen atoms (e.g., chloride, bromide, fluoride, or iodide groups), carboxylic acids (e.g., C(O)OH), alcohols (e.g., hydroxyl, OH), esters (e.g., C(O)OR or OC(O)R), aldehydes (e.g., C(O)H), carbonyls (e.g., represented by C(O)R, or alternatively C=O), acyl halides (e.g., C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (e.g., OC(O)OR), alkoxy (e.g., OR), acetals (e.g., C(OR)R"", where each OR is an alkoxy group, which may be the same or different, and R"", is an alkyl or alkenyl group), phosphates (e.g., P(O)4 3 ), thiol (e.g., SH), sulfoxide (e.g., S(O)R), sulfinic acid (e.g., S(O)OH), sulfonic acid (e.g., S(O)2OH), thial (e.g., C(S)H), sulfate (e.g., S(O)4 2 ), sulfonyl (e.g., S(O)2), amido (e.g., C(O)NR2, or N(R)C(O)R), azido (e.g., N3), nitro (e.g., NO2), cyano (e.g., CN), isocyano (e.g., NC), acyloxy (e.g., OC(O)R), amino (e.g., NR2, NRH, or NH2), carbamoyl (e.g., OC(O)NR2, OC(O)NRH, or OC(O)NH2), sulfonamide (e.g., S(O)2NR2, S(O)2NRH, S(O)2NH2, N(R)S(O)2R, N(H)S(O)2R, N(R)S(O)2H, or N(H)S(O)2H), alkyl groups, alkenyl groups, and cyclyl (e.g., carbocyclyl or heterocyclyl) groups. As defined herein, R is an alkyl, alkenyl, or alkynyl group.

[0420] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the foregoing description, but is instead set forth in the appended claims.

[0421] Where ranges are given, the endpoints are included. Additionally, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can be understood to refer to any particular value or subrange within a stated range in different embodiments of the present disclosure to one tenth of the unit of the lower limit of that range, unless the context clearly indicates otherwise.

[0422] In addition, it should be understood that any particular embodiment of the present disclosure within the prior art may be expressly excluded from any one or more of the claims. Such an embodiment may be excluded even if the exclusion is not expressly stated herein, since it is considered to be known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any nucleic acid or protein encoded thereby, any method of production, any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0423] It will be further understood that certain features that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0424] All cited sources, e.g., references, publications, databases, database entries, and art cited herein are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements in the cited sources and this application, the statements in this application shall control.

[0425] The section and table headings are not intended to be limiting. EXAMPLES

[0426] Example 1 Generation of empty lipid nanoparticles Empty lipid nanoparticles were prepared according to the process outlined in Figure 1. Lipids (ionized lipid:DSPC:cholesterol:DMG-PEG2000 lipid) were dissolved in ethanol at a total concentration of 24 mg / mL and mixed with acidification buffer (45 mM acetate buffer at pH 4). The lipid solution and acidification buffer were mixed using a multi-inlet vortex mixer at a lipid:buffer volume ratio of 3:7 for mixer 1 and mixer 2, and at a lipid:buffer (25% ethanol) volume ratio of 1:3 for mixer 3. After a dwell time of 5 seconds, the resulting eLNPs were mixed with 55 mM sodium acetate at pH 5.6 at a eLNP:buffer volume ratio of 5:7. The resulting diluted eLNPs were then buffer exchanged and concentrated using tangential flow filtration (TFF) to a final buffer containing 5 mM sodium acetate at pH 5.0. A 70% sucrose solution in 5 mM acetate buffer at pH 5 was then added subsequently.

[0427] Example 2 Grain size comparison Lipids (ionized lipid:DSPC:cholesterol:DMG-PEG2000 lipid) were dissolved in ethanol at a concentration of 24 mg / mL (40 mM in total) and mixed with acidification buffer (37.5 mM acetate buffer at pH 4 for sample no. 1 and 37.5 mM acetate buffer at pH 5 for sample no. 2).

[0428] The lipid solution was delivered at 2.5 mL / min and the acidified aqueous buffer stream was delivered at 7.5 mL / min. These two streams were mixed using a mixing T-tube with an internal diameter of 0.5 mm. The lipid concentration after nanoprecipitation was 6 mg / mL. 600 mL of LNP solution was generated for each sample.

[0429] For sample number 1 a 30 kDa mPES filter was used and for sample number 2 a 100 kDa mPES filter was used. A 5-fold volume ultrafiltration (UF1) was performed first, followed by a 5-fold volume diafiltration (DF) against a 30 kDa filter or an 8-fold volume diafiltration (DF) against a 100 kDa filter. The final step was another 8-fold ultrafiltration (UF2).

[0430] See Table 2-A for final lipid concentrations and calculated yields for each sample.

[0431] After TFF, a 70% sucrose solution in 37.5 mM acetate buffer at pH 4 (for sample 1) or pH 5 (for sample 2) was added to produce a final product of 74.5 mg / mL LNP and 200 mg / mL sucrose. [Table 1]

[0432] The average diameter of the empty lipid nanoparticles prepared as described above at pH 4 and pH 5 was measured by dynamic light scattering (DLS). The diameters are given in nanometers (nm) and are shown in Table 2-B. As can be seen from the data, nanoprecipitation at pH 4 results in smaller sized particles compared to pH 5. [Table 2]

[0433] Example 3 Characterization of empty lipid nanoparticles The average size of empty lipid nanoparticles prepared according to the process of Example 2, with the modifications noted below, was measured.

[0434] The average size of empty lipid nanoparticles was compared at different pH values ​​and different buffer concentrations. The average particle size was measured by dynamic light scattering (DLS). The results are shown in Figure 2, which shows that low pH and high buffer concentration favor the formation of small size particles.

[0435] The average size of empty lipid nanoparticles was compared at different buffer strengths and different lipid solution concentrations. The average particle size was measured by DLS. The results are shown in Figure 3, which shows that high buffer concentrations favor the formation of small sized particles.

[0436] The average size of empty lipid nanoparticles was compared over a period of 25 hours at various buffer strengths (20 mM, 37.5 mM, 75 mM, and 120 mM). The average particle size was measured by DLS. The results are shown in Figure 4 and indicate that high buffer concentrations favor the formation of small sized particles that remain small over a period of 25 hours.

[0437] The average size of empty lipid nanoparticles was compared over a period of 25 hours at various pH values. The average particle size was measured by DLS. The results are shown in FIG. 5 and indicate that low pH favors the formation of small sized particles that remain small over a period of 25 hours.

[0438] The zeta potential of the empty lipid nanoparticles prepared according to the process of Example 2 was measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential by the principle of "massively parallel phase analysis light scattering" or MP-PALS. This measurement is more sensitive and induces less stress than ISO method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. The results are shown in Figure 6, which show high zeta potential at low pH, which is almost independent of buffer concentration and lipid solution concentration.

[0439] The composition of the empty lipid nanoparticles was assessed by cryo-EM and the results are shown in Figure 7. LNPs precipitated at pH 4 show smaller size and more uniform composition compared to those prepared at pH 5.

[0440] Example 4 Preparation of loaded lipid nanoparticles Empty lipid nanoparticles prepared according to Example 1, Figure 1, were loaded with nucleic acid (mRNA) according to the process shown in Figure 8. Loading of mRNA was performed using a post-loading (PHL) process. eLNPs at a lipid concentration of 11.72 mg / mL in 5 mM acetate (pH 5) and 75 g / L sucrose were mixed with mRNA at a concentration of 1.0 mg / mL in 42.5 mM sodium acetate at pH 5.0. The eLNP solution and mRNA were mixed in a 3:2 eLNP:mRNA volume ratio using a multi-inlet vortex mixer mixer. Once the eLNPs were loaded with mRNA, after a 60 second residence time, they were mixed in-line with a neutralization buffer containing 120 mM TRIS (pH 8.12) in a 5:1 nanoparticle:buffer volume ratio. After this addition step, the nanoparticle formulation was in-line mixed with a buffer solution containing 20 mM TRIS (pH 7.5), 1.42 mg / mL DMG-PEG2000, and 2.5 mg / mL GL-67 (sterolamine) at a nanoparticle:buffer volume ratio of 6:1. The resulting nanoparticle suspension was concentrated using tangential flow filtration (TFF) and diluted in a running buffer (20 mM TRIS, 14.3 mM sodium acetate, and 32 g / L sucrose, pH 7.5) containing 300 nM NaCl solution to a final buffer matrix containing 70 mM NaCl. The resulting nanoparticle suspension was filtered through a 0.8 / 0.2 μm capsule filter and filled into glass vials at an mRNA concentration of approximately 1 mg / mL (e.g., 0.5-2 mg / mL).

[0441] Example 5 Characterization of loaded lipid nanoparticles Loaded lipid nanoparticle compositions were prepared at various mRNA stock concentrations following the process described in Example 4. For process details, see Table 5-A below. [Table 3]

[0442] The resulting mean particle size (measured by DLS) and polydispersity (PDI) values ​​are compared in Figure 9. Values ​​were calculated using cumulant analysis. The diameter of the MP columns was measured after the PI buffer step. The mean particle size was consistently between 75 and 85 nm throughout. The encapsulation efficiency was >98%. The loading mRNA concentration had little effect on the particle size.

[0443] Example 6 Alternative generation of empty lipid nanoparticles Empty lipid nanoparticles were prepared according to the process outlined in Figure 11. Lipids (ionized lipid:DSPC:cholesterol:DMG-PEG2000 lipid) were dissolved in ethanol at a concentration of 24 mg / mL and mixed with acidified buffer (37.5 mM acetate buffer at pH 4). After a 5 second residence time, the resulting eLNPs were mixed with 37.5 mM sodium acetate at pH 4 in an eLNP:buffer volume ratio of 5:7. The resulting diluted eLNPs were then buffer exchanged and concentrated using tangential flow filtration (TFF) to a final buffer containing 37.5 mM sodium acetate at pH 4. A 70% sucrose solution in 37.5 mM acetate buffer at pH 4 was then subsequently added.

[0444] Example 7 Alternative preparation of loaded lipid nanoparticles Empty lipid nanoparticles prepared according to Example 6, Figure 11, were loaded with nucleic acid (mRNA) according to the process shown in Figure 12. Loading of mRNA was performed using a post-loading (PHL) process. mRNA in 32.5 mM acetate at pH 5 was added to water using dialysis. Concentration was measured using NaOH digestion. Buffers were used to determine the concentration of acetic acid and sodium acetate for 37.5 mM acetate buffer at pH 4, 4.5, 5, 5.5 or 6. This additional concentrated buffer was used to dilute the mRNA with water (with additional water) to 1.6 mg / mL of mRNA in 37.5 mM buffer at the respective pH. eLNP solutions at a lipid concentration of 37.25 mg / mL in 37.5 mM acetate and 20% sucrose at pH 4, 4.5, 5, 5.5 or 6 were mixed with mRNA at a concentration of 1.6 mg / mL in 37.5 mM sodium acetate at the same pH as the eLNP solution. The eLNP solution and mRNA were mixed at a volumetric ratio of 1:2.5 eLNP:mRNA using a multi-inlet vortex mixer. Once the eLNPs were loaded with mRNA, after a 60 second residence time, they were mixed in-line with a neutralization buffer containing TRIS buffer and 32.3% sucrose. After this addition step, the nanoparticle formulation was mixed in-line with a buffer containing 20 mM TRIS (pH 7.5), 4.5 mg / mL DMG-PEG2000.

[0445] Example 8 Characterization of loaded lipid nanoparticles – particle size Table 8-A shows the encapsulation efficiency of loaded lipid nanoparticles prepared according to Example 7, Figure 12. Figure 13 shows the average diameter in nm of empty lipid nanoparticles and loaded lipid nanoparticles at different loading pH of mRNA solution measured before mixing. Pre-neutra or Pre-neu nanoparticles are nanoparticles that have not been subjected to neutralization buffer. Post-neutra or Post-neu nanoparticles are nanoparticles that have been subjected to neutralization buffer. [Table 4]

[0446] Table 8-B shows a comparison of the encapsulation efficiency of loaded lipid nanoparticles prepared using procedures similar to those outlined in Examples 6 and 7, but where the acidified buffer had a pH of 5. Figure 14 shows the average diameter in nm of the loaded lipid nanoparticles at different loading pHs of the empty lipid nanoparticles and mRNA solutions measured before mixing. [Table 5]

[0447] N:P is the ratio of nitrogen to phosphorus in the nanoparticles.

[0448] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Claims

1. A process for preparing an empty lipid nanoparticle composition, comprising: (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) mixing a lipid solution containing a PEG-lipid with a buffered aqueous solution having a pH of about 4.5 or less, wherein the buffered aqueous solution has a buffer concentration of about 30 mM or more and / or the buffered aqueous solution has an ionic strength of about 15 mM or less.

2. The process according to claim 1, wherein the process produces an empty lipid nanoparticle composition characterized by a zeta potential of about 35 mV or more.

3. The process according to claim 1 or 2, wherein the lipid solution has a lipid concentration of about 5 to about 100 mg / mL.

4. The process according to claim 1 or 2, wherein the empty lipid nanoparticle composition comprises empty lipid nanoparticles having an average diameter of about 30 nm or less.

5. The process according to claim 1 or 2, wherein the empty lipid nanoparticle composition comprises empty lipid nanoparticles having an average diameter that increases by less than about 150% over 25 hours.

6. One or more additional steps selected from the group consisting of: diluting the composition with a dilution buffer, adjusting the pH of the composition to a pH of about 5 to about 6, filtering the composition, concentrating the composition, exchanging the buffer of the composition, and adding a cryoprotectant to the composition.

7. An empty lipid nanoparticle composition prepared by the process according to claim 1.

8. A process for preparing a loaded lipid nanoparticle composition, comprising: (a) mixing a lipid solution containing (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid with a buffered aqueous solution having a pH of less than about 4.5 to obtain an empty lipid nanoparticle composition; and (b) combining the empty lipid nanoparticle composition with a payload to form the loaded lipid nanoparticle composition.

9. The process according to claim 8, wherein the payload comprises a nucleic acid, and the nucleic acid is prepared as a nucleic acid solution comprising (i) the nucleic acid and (ii) a buffer capable of maintaining an acidic pH.

10. One or more additional steps selected from the group consisting of: diluting the composition with a dilution buffer, adjusting the pH of the composition to a pH of about 7 to about 8. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The step of filtering the composition, The step of concentrating the composition, The step of exchanging the buffer of the composition, The step of adding a surfactant to the composition, and The step of adding an osmotic pressure regulator to the composition The process according to claim 8 or 9, further comprising one or more additional steps selected from

11. (c) adjusting the pH of the composition to a pH of from about 7 to about 8; (d) adding one or more surfactants to the composition; (e) concentrating the composition; (f) adding an osmotic pressure regulator to the composition; (g) diluting the composition The process according to claim 8, further comprising

12. A filled lipid nanoparticle composition prepared by the process according to claim 8 or 9.

13. The following components: (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) an empty lipid nanoparticle composition comprising an empty lipid nanoparticle containing a PEG-lipid, The empty lipid nanoparticle composition, (a) substantially free of payload, (b) having a pH of from about 3 to about 5, (c) characterized by a zeta potential of about 35 mV or more, The empty lipid nanoparticle composition.

14. The following components: (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, (iv) a PEG-lipid, and (v) a filled lipid nanoparticle composition comprising a filled lipid nanoparticle containing a payload, The filled lipid nanoparticle composition, having a pH of from about 4.5 to about 8, the filled lipid nanoparticle composition.

15. The filled lipid nanoparticle composition according to claim 14, wherein the concentration of the payload is from about 0.1 to about 10 mg / mL.

16. The ionizable lipid comprises a compound of formula (I): 【Chemical 1】 or an N-oxide or salt thereof, wherein R １ is 【Chemical 2】 is [Chemical Formula 3] represents a point of attachment and is selected from R ａα 、R ａβ 、R ａγ 、and R ａδ are each independently selected from H, C ２～１２ alkyl, and C ２～１２ alkenyl, R ２ and R ３ are each independently selected from C １～１４ alkyl and C ２～１４ alkenyl, R ４ is —(CH ２ ) ｎ OH and 【Chemical Formula 4】 n is selected from 1, 2, 3, 4, and 5, represents a point of attachment, 【Chemical Formula 5】 n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, R １０ is N(R) ２ and Each R is independently C １～６ alkyl, C ２～３ alkenyl, and H, and is selected from M and M' are each independently selected from -C(O)O- and -OC(O)-, Each R ５ is independently selected from C １～３ alkyl, C ２～３ alkenyl, and H Each R ６ is independently selected from C １～３ alkyl, C ２～３ alkenyl, and H, l is selected from 1, 2, 3, 4, and 5, R' is C １～１２ alkyl or C ２～１２ alkenyl, and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, the process according to any one of claims 1, 2, 8, 9, and 11 or the lipid nanoparticle composition according to any one of claims 7, 13, 14, and 15.

17. The phospholipid is ​ 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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-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-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lysophosphatidylcholine), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-Diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 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), and sphingomyelin The process according to any one of claims 1, 2, 8, 9 and 11 or the lipid nanoparticle composition according to any one of claims 7, 13, 14 and 15, selected from

18. The structural lipid is selected from cholesterol, fucosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, alpha-tocopherol, hopanoid, phytosterol, steroid, or a mixture thereof, the process according to any one of claims 1, 2, 8, 9 and 11 or the lipid nanoparticle composition according to any one of claims 7, 13, 14 and 15.

19. The PEG-lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof, the process according to any one of claims 1, 2, 8, 9 and 11 or the lipid nanoparticle composition according to any one of claims 7, 13, 14 and 15.

20. The lipid solution, the empty lipid nanoparticle composition, or the filled lipid nanoparticle composition, respectively, with respect to the total lipid about 40 mol% to about 50 mol% of ionizable lipid, about 10 mol% to about 12 mol% of phospholipid, about 37 mol% to about 42 mol% of structural lipid, and about 0.25 mol% to about 0.75 mol% of PEG-lipid, the process according to any one of claims 1, 2, 8, 9 and 11 or the lipid nanoparticle composition according to any one of claims 7, 13, 14 and 15.

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