Process for preparing lipid nanoparticle compositions for delivery of payload molecules to airway epithelia - Patent Application 20070233633

Abstract

Provided are lipid nanoparticle compositions, and processes for their preparation, useful for delivering therapeutic or prophylactic agents to the respiratory epithelium of a patient.

Description

[Technical field]

[0001] Provided are lipid nanoparticle compositions for delivery of therapeutic or prophylactic agents to the respiratory epithelium of a patient, and processes for their preparation. [Background technology]

[0002] Respiratory epithelial cells line the airways. Their main function is to humidify the airways, protect the airway tract from potential pathogens, infections, and tissue damage, and / or facilitate gas exchange. Dysfunction of airway epithelial cells can lead to many disorders, including, for example, asthma and chronic obstructive pulmonary disease (COPD).

[0003] There is a need for improved therapies for treating disorders associated with dysfunction of airway epithelial cells or other disorders that benefit from delivery of nucleic acid molecules or other payload molecules to airway epithelial cells.Furthermore, the effective targeted delivery of payload molecules to airway epithelial cells represents a continuing medical challenge.Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and prophylactic agents, such as nucleic acids, to airway epithelial cells. Summary of the Invention

[0004] The present disclosure provides LNP molecules for delivering nucleic acid molecules, such as mRNA therapeutics, for prophylactic effect in patients.

[0005] In one embodiment, provided herein is a process for preparing a loaded lipid nanoparticle composition comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid with an aqueous buffer solution having a pH of about 4.5 or less to obtain a blank lipid nanoparticle composition; (b) mixing the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition, wherein the payload is for delivery to an epithelial cell; and (c) adding a cationic drug to the loaded lipid nanoparticle composition.

[0006] In one aspect, provided herein is a lipid nanoparticle composition prepared by the processes disclosed herein.

[0007] In one aspect, provided herein is a method of delivering a payload to a cell, the method comprising contacting the cell with a lipid nanoparticle composition disclosed herein.

[0008] In one aspect, provided herein is a method of treating or preventing a disease in a patient, the method comprising administering to the patient a lipid nanoparticle composition disclosed herein.

[0009] Each limitation may encompass various embodiments. Thus, each limitation involving any one element or combination of elements is anticipated to be 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 are possible and can be practiced or carried out in various ways. [Brief description of the drawings]

[0010] [Figure 1] A general process for preparing empty LNPs (eLNPs) is shown, where nanoprecipitation is performed at pH 4 followed by titration to pH 5.

[0011] [Diagram 2] The effect of pH and lipid solution concentration on the mean diameter (nm) of eLNPs is shown.

[0012] [Diagram 3]The effect of buffer concentration and lipid solution concentration on the average diameter (nm) of eLNPs is shown.

[0013] [Figure 4] The effect of pH and buffer concentration on the mean diameter (nm) of eLNPs is shown.

[0014] [Diagram 5] 1 shows the effect of pH on the mean diameter (nm) of eLNPs over time.

[0015] [Figure 6] Zeta potential (mV) of eLNPs prepared with various concentrations of lipid solution (LSS) in 37.5 mM acetate buffer (left) or 75 mM acetate buffer (right) as a function of pH.

[0016] [Figure 7] Cryo-EM images of eLNPs precipitated at pH 4 (left) and pH 5 (right) are shown.

[0017] [Figure 8] A general process for preparing loaded LNPs (fLNPs) is shown, where encapsulation is performed at pH 5.

[0018] [Figure 9] FIG. 9 shows the mean particle size and polydispersity index (PDI) of fLNPs prepared according to the process of FIG.

[0019] [Figure 10] 1 shows capillary zone electrophoresis plots of eLNPs prepared at pH 4 and eLNPs prepared at pH 5 run using acetate buffer at pH 5.

[0020] [Figure 11] Another general process for preparing empty LNPs (eLNPs) is shown, where nanoprecipitation is performed at pH 4.

[0021] [Figure 12] Another general process for preparing loaded LNPs (fLNPs) is shown, where encapsulation is performed at pH 4-pH 6.

[0022] [Figure 13] The effect of pH on the mean diameter (nm) of fLNPs is shown.

[0023] [Figure 14] 1 shows the effect of pH on the mean diameter (nm) of fLNPs prepared at pH 5.

[0024] [Figure 15] Another general process for preparing loaded LNPs (fLNPs) is shown, where encapsulation is performed from pH 5.

[0025] [Figure 16] Another general process for preparing loaded LNPs (fLNPs) is shown, where encapsulation is performed from pH 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Provided are, inter alia, lipid nanoparticle (LNP) compositions and their preparation processes comprising a payload for delivery to the respiratory epithelium of a patient, the lipid nanoparticle compositions being prepared by loading an empty lipid nanoparticle composition having certain advantageous properties prepared as described herein. In particular, some embodiments include an empty nanoparticle composition for preparing lipid nanoparticles having a substantially uniform morphology and a small average particle size with a size distribution and a relatively high zeta potential, which are loaded with a payload. The empty lipid nanoparticle composition may be formed under conditions favoring a relative uniformity and small size that may remain stable for extended periods of time even in the presence of ethanol, facilitating work-up and manipulation. In particular, the empty lipid nanoparticles may 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 composition in post-loading (PHL) with nucleic acids or other therapeutic agents, providing a therapeutic loaded lipid nanoparticle (fLNP) composition for delivery to cells, e.g., the respiratory epithelium, of a patient for the treatment or prevention of disease.

[0027] Process for preparing empty lipid nanoparticle compositions The process of preparing the empty lipid nanoparticle composition may include nanoprecipitation of empty lipid nanoparticles at low pH, low ionic strength, high buffer strength, or a combination thereof. Nanoprecipitation is a unit operation that allows LNPs to self-assemble from their individual lipid components by dynamic mixing followed by aging and continuous dilution. This unit operation may include three individual steps: mixing of aqueous and organic inputs, aging of LNPs, and dilution after a controlled residence time. These steps are considered as one unit operation due to their continuous nature. The unit operation includes continuous in-line mixing of three liquid streams and one in-line aging step, namely, mixing of an aqueous buffer solution with a lipid stock solution, aging through a controlled residence time, and dilution of the nanoparticles. The nanoprecipitation itself takes place in a mixer of suitable size designed to allow continuous high energy mixing of an aqueous buffer solution with a lipid stock solution dissolved in ethanol. Both the aqueous buffer solution and the lipid stock solution flow simultaneously into the mixing device continuously throughout the operation. The ethanol content that keeps the lipids dissolved is suddenly reduced and the lipids all precipitate out of each other. Thus, the particles self-assemble in the mixing chamber.

[0028] One of the objectives of the unit operation is to exchange the solution into a completely aqueous buffer solution that is free of ethanol to achieve the target concentration of LNPs, which may be achieved first by reaching a target processing concentration, then diafiltration, and then (if necessary) a final concentration step after the ethanol has been completely removed.

[0029] For example, some embodiments include a process for preparing an empty lipid nanoparticle composition, the process comprising: (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid with an aqueous buffer solution having a pH of about 4.5 or less.

[0030] In some embodiments, the aqueous buffer solution has a pH of about 3.5 to about 4.5. In further embodiments, the aqueous buffer solution has a pH of about 4.

[0031] The process of preparing the empty lipid nanoparticle composition may include precipitating the nanoparticles at a relatively high buffer concentration, for example, a concentration high enough to affect the buffering effect of lipids in the lipid solution. In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 30 mM. In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 40 mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 30 mM to about 100 mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 40 mM to about 75 mM. In further embodiments, the aqueous buffer solution has a buffer concentration of about 33 mM, about 37.5 mM, or about 45 mM.

[0032] 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.

[0033] In some embodiments, the aqueous buffer solution used in the process of preparing the empty lipid nanoparticles has a pH lower than the pKa of the resulting empty lipid nanoparticles.

[0034] The process of preparing the lipid nanoparticle composition may further comprise precipitating the nanoparticles at a relatively low ionic strength.For example, the aqueous buffer 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 aqueous buffer 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.

[0035] Suitable buffers include buffers that support an acidic pH, for example, pH 3-5. In some embodiments, the aqueous buffer solution may include an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the aqueous buffer solution includes an acetate buffer or a citrate buffer. In further embodiments, the aqueous buffer solution is an acetate buffer, for example, a sodium acetate buffer.

[0036] The process for preparing the empty lipid nanoparticle composition may include precipitating the 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 at a pH range of 3-6, at least about 33% of the maximum zeta potential achievable for the composition at a pH range of 3-6, at least about 50% of the maximum zeta potential achievable for the composition at a pH range of 3-6, at least about 66% of the maximum zeta potential achievable for the composition at a pH range of 3-6, or at least about 75% of the maximum zeta potential achievable for the composition at a pH range of 3-6.

[0037] Zeta potential is a measure of the electrokinetic potential in 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 Mobius zeta potential instrument from Wyatt Technologies. 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 composition described herein is measured using an instrument that employs the principle of MP-PALS.

[0038] The process can further use a lipid solution, which is a composition comprising at least four lipid components: an ionic lipid, a phospholipid, a structured lipid, and a PEG-lipid. Any suitable concentration of lipid solution can be used. For example, the lipid solution can 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.

[0039] The lipid solution may further comprise an organic solvent, such as an alcohol, such as ethanol. The organic solvent may 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 by volume, or more than 95% ethanol by volume.

[0040] 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 % ionic lipid relative to total lipid.

[0041] In some embodiments, the lipid solution comprises about 5 mol % to about 15 mol %, about 8 mol % to about 13 mol %, or about 10 mol % to about 12 mol % phospholipid based on total lipid.

[0042] In some embodiments, the lipid solution 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 based on total lipid.

[0043] 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 mol% to about 0.75 mol% PEG-lipid relative to total lipid.

[0044] In some embodiments, the lipid solution comprises, relative to total lipid, the following: about 40 mol % to about 50 mol % of an ionic lipid; about 10 mol% to about 12 mol% of phospholipids, about 37 mol % to about 42 mol % of structural lipids, and About 0.25 mol% to about 0.75 mol% PEG-lipid.

[0045] In some embodiments, the lipid solution comprises, relative to total lipid, the following: Approximately 49 mol% ionic lipids, about 11 mol% to about 12 mol% of phospholipids, Approximately 39 mol% structural lipids, and Approximately 0.5 mol% PEG-lipid.

[0046] Upon mixing the lipid solution with the buffer solution, lipid nanoparticles are precipitated to prepare an empty lipid nanoparticle composition. Precipitation can be performed by ethanol drop precipitation, for example, by using a high energy mixer (e.g., T-junction, confined impinging jet, microfluidic mixer, vortex mixer) to controllably introduce lipids (in ethanol) into a suitable anti-solvent (i.e., water) to drive liquid supersaturation and spontaneous precipitation into lipid particles. 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.

[0047] 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). In addition, 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 comparing cryo-EM images of empty lipid nanoparticles. The image on the left shows particles prepared at pH 4 according to the present disclosure having a uniform morphology, in contrast to the image on the right, in which particles were prepared at pH 5.

[0048] The precipitated empty lipid nanoparticles are substantially free of payload, where payload refers to any therapeutic or prophylactic agent, e.g., a polypeptide or nucleic acid, intended for delivery to a cell.

[0049] 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 over time. For example, the average diameter of the empty lipid nanoparticles increases less than about 150% in 25 hours, or increases less than about 100% in 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. The stability is demonstrated in an empty lipid nanoparticle composition comprising an organic solvent, such as an 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.

[0050] In some embodiments, the mean diameter of the lipid nanoparticles remains less than 50 nm for 25 hours at 25° C., or remains less than 40 nm for 25 hours at 25° C. In some embodiments, the mean diameter of the lipid nanoparticles remains less than 30 nm for at least 24 hours in the presence of 25% ethanol by volume. In some embodiments, the mean diameter of the lipid nanoparticles remains less than 30 nm for at least 24 hours in the presence of 25% ethanol by volume at 25° C.

[0051] In some embodiments, the empty lipid nanoparticle composition is contained in a storage solution. In some embodiments, the storage solution includes 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 included in the storage solution includes 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, e.g., 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.

[0052] In some embodiments, the storage solution comprises a cryoprotectant. In some embodiments, the cryoprotectant is one or more cryoprotectants, e.g., a polyol (e.g., a diol or triol, e.g., 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 sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 2 ... 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. 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 and sodium chloride.

[0053] In some embodiments, the concentration of empty lipid nanoparticles in the storage solution is about 5 to about 100 mg / mL, about 15 to about 75 mg / mL, or about 20 to about 60 mg / mL.

[0054] 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.

[0055] In some embodiments, the average diameter of the empty lipid nanoparticles contained in the storage solution remains less than 30 nm for at least 4 months at 5°C.

[0056] 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 about pH 5 to about 6; filtering the composition; concentrating the composition; exchanging the buffer of the composition; and Adding a cryoprotectant to the composition.

[0057] In some embodiments, the process of preparing the empty lipid nanoparticle composition can further comprise one, two, three, four, five or all of the steps listed above. Some steps can be repeated. The steps can be, but do not have to be, performed in the order listed above. Each of the steps refers to an action related to the composition obtained from the step performed before. For example, if the process comprises a step of exchanging the buffer of the composition, the buffer exchange is performed on the composition obtained from the previous step, and in this case, the previous step can be any of the steps listed above.

[0058] 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 with 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 further embodiments, the dilution buffer is an acetate buffer, e.g., 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.

[0059] For example, diluting the composition with a dilution buffer can correspond to the ILD buffer step in FIG.

[0060] In some embodiments, the empty lipid nanoparticle composition undergoes controlled residence time aging prior to dilution of the nanoparticles, in some embodiments, the residence time is about 1 to about 30 seconds, about 2 to about 15 seconds, about 3 to about 10 seconds, about 4 to about 7 seconds, or about 5 seconds.

[0061] In some embodiments, the process includes adjusting the pH of the composition to about pH 5 to about 6. For example, if the empty lipid nanoparticle composition underwent 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.

[0062] For example, adjusting the pH of the composition to about pH 5 to about 6 may correspond to the ILD buffer step in FIG.

[0063] In some embodiments, the process does not include adjusting the pH of the composition. For example, if the blank lipid nanoparticle composition underwent nanoprecipitation at pH 4, the pH of the composition is maintained at about 4.

[0064] 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 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 further embodiments, the buffer for adjusting the pH is an acetate buffer, for example, sodium acetate. In some embodiments, the pH of the buffer for adjusting the pH 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 for adjusting the pH can also be a dilution buffer.

[0065] In some embodiments, the process comprises adding a cryoprotectant to the composition. In some embodiments, the cryoprotectant is one or more cryoprotectants, e.g., a polyol (e.g., a diol or triol, e.g., 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 sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 2 ... 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant is sucrose.

[0066] 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 included in the cryoprotectant solution includes 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 further embodiments, the buffer is an acetate buffer, e.g., 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.

[0067] 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.

[0068] 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% w / v sucrose.

[0069] For example, adding a cryoprotectant to the composition may correspond to the sucrose spiking step of FIGS.

[0070] In some embodiments, the process includes any 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 exchanging steps may be accomplished using tangential flow filtration (TFF).

[0071] For example, filtering the composition, concentrating the composition, and exchanging the buffer of the composition can correspond to the TFF steps of FIGS.

[0072] In some embodiments, the filtration step may remove organic solvents (e.g., alcohols such as ethanol) and other unwanted components from the lipid nanoparticle composition.

[0073] 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, to about 1 mM to about 10 mM, about 2 mM to about 8 mM, about 4 mM to about 6 mM, or about 5 mM. In some embodiments, the buffer exchange step includes removing or reducing the amount of organic solvent.

[0074] In some embodiments, the concentrating step may increase the concentration of empty lipid nanoparticles in the composition.

[0075] 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.

[0076] 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.

[0077] In some embodiments, the process comprises at least two steps: adjusting the pH of the composition to about pH 5 to about 6, and adding a cryoprotectant to the composition.

[0078] In some embodiments, the process comprises at least two steps: adjusting the pH of the composition to about pH 4.5 to about 6, and adding a cryoprotectant to the composition.

[0079] 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.

[0080] In some embodiments, the process includes diluting the composition.

[0081] 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 further embodiments, the dilution buffer is an acetate buffer, e.g., 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.

[0082] 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 diluting the composition is about 4, about 4.5, about 5, about 5.5, or about 6.

[0083] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids with an aqueous buffer solution having a pH of about 4; (b) optionally adjusting the pH of the solution from the previous step by adding a pH adjusting buffer of about pH 5 to about 6; (c) optionally filtering the solution from the previous step to reduce its buffer concentration; (d) optionally adding sucrose to the solution from the previous step; and (e) Optionally, diluting the solution from the previous step.

[0084] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids with an aqueous buffer solution having a pH of about 4; (b) adjusting the pH of the solution from the previous step by adding a pH adjusting buffer to a pH of about 5 to about 6; (c) optionally filtering the solution from the previous step to reduce its buffer concentration; (d) adding sucrose to the solution from the previous step; and (e) Optionally, diluting the solution from the previous step.

[0085] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids with an aqueous buffer solution having a pH of about 4; (b) optionally adding sucrose to the solution from the previous step; and (c) Optionally, diluting the solution from the previous step.

[0086] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids with an aqueous buffer solution having a pH of about 4; (b) adding sucrose to the solution from the previous step; and (c) Optionally, diluting the solution from the previous step.

[0087] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids with an aqueous buffer solution having a pH of about 4; (b) adjusting the pH of the composition to about pH 5 to about 6; (c) filtering the composition; (d) concentrating the composition; (e) exchanging the buffer of the composition; and (f) adding a cryoprotectant to the composition.

[0088] In some embodiments, the empty lipid nanoparticle composition may be prepared by a process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid mixing a lipid solution comprising the lipids 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; (e) exchanging the buffer of the composition; and (f) adding a cryoprotectant to the composition.

[0089] Some embodiments include an empty lipid nanoparticle composition prepared by any of the processes described herein.

[0090] Process for Preparing Loaded Lipid Nanoparticle Compositions The empty lipid nanoparticle composition can be used to prepare a filled lipid nanoparticle (fLNP) composition by mixing the empty lipid nanoparticle composition with a payload.

[0091] Some embodiments include a process for preparing a filled lipid nanoparticle composition, the process comprising mixing an empty lipid nanoparticle composition, e.g., prepared by any of the processes described above, with a payload to form the filled lipid nanoparticle composition.

[0092] Some embodiments include a process for preparing a loaded lipid nanoparticle composition, the process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid with an aqueous buffer solution having a pH of about 4.5 or less to obtain a blank lipid nanoparticle composition; (b) mixing the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition, wherein the payload is for delivery to an epithelial cell; and (c) adding a cationic drug to the loaded lipid nanoparticle composition.

[0093] In some embodiments, the mixing in step (b) is performed at a pH of about 4.5 to about 5.5. In some embodiments, the mixing 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 mixing the empty lipid nanoparticle composition with a payload. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 5 prior to mixing the empty lipid nanoparticle composition with a payload.

[0094] In some embodiments, the mixing is performed at a pH of about 4 to about 6. In some embodiments, the mixing 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 mixing the empty lipid nanoparticle composition with a 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 mixing the empty lipid nanoparticle composition with a payload.

[0095] In some embodiments, the payload comprises a nucleic acid, e.g., an mRNA, which in some embodiments encodes a protein expressed in airway epithelium, e.g., CFTR, short palate lung and nasal epithelium clone 1 (SPLUNC1), and alpha-1-antitrypsin (AAT).

[0096] The nucleic acid payload can be provided as a nucleic acid solution comprising (i) a nucleic acid, e.g., DNA or RNA (e.g., mRNA), and (ii) a buffer capable of maintaining an acidic pH, e.g., 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.

[0097] In some embodiments, the pH of the nucleic acid solution is about 4, about 4.5, about 5, about 5.5, or about 6.

[0098] 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 further embodiments, the buffer is an acetate buffer, for example, a sodium acetate buffer. The buffer concentration of the nucleic acid solution can 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.

[0099] 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.

[0100] In some embodiments, the buffer concentration is about 20 mM to about 32 mM, about 22 mM to about 30 mM, or about 24 mM to about 28 mM. In some embodiments, the buffer concentration is about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM. In some embodiments, the buffer concentration is about 26 mM.

[0101] The nucleic acid solution may contain nucleic acid at a 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] The empty lipid nanoparticle composition is mixed with a nucleic acid solution to post-load the empty lipid nanoparticles with nucleic acid. A high-energy mixer (e.g., a T-junction, a confined impinging jet, a microfluidic mixer, a vortex mixer) can 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 of, 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.

[0106] Encapsulation efficiency is advantageously high for the empty lipid nanoparticle compositions disclosed herein. Encapsulation efficiency (EE) indicates the amount of therapeutic and / or prophylactic agent that is encapsulated or associated with lipid nanoparticles after preparation relative to the amount initially provided. The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent (e.g., payload) contained in a solution containing the lipid nanoparticles before and after the lipid nanoparticles are destroyed by one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) contained in the solution. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) contained in the solution. In the lipid nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent 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.

[0107] In some embodiments, the cationic agent solution has a pH of about 7 to about 8, or about 7.5.

[0108] In some embodiments, the buffer concentration of the cationic agent solution is about 5 mM to about 100 mM, about 5 mM to about 50 mM, about 10 mM to about 30 mM, or about 20 mM.

[0109] In some embodiments, the buffer of the cationic drug solution is an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffer of the cationic drug solution comprises Tris.

[0110] In some embodiments, the concentration of the cationic drug in the cationic drug solution is about 0.1 to about 50 mg / mL, about 1 to about 30 mg / mL, about 1 to about 10 mg / mL, or about 2 to about 3 mg / mL.

[0111] In some embodiments, the cationic drug in the cationic drug solution has a concentration of about 0.08 to about 1.3 mg / mL, about 0.1 to about 1.1 mg / mL, about 0.3 to about 0.9 mg / mL, or about 0.5 to about 0.7 mg / mL. In some embodiments, the cationic drug in the cationic drug solution has a concentration of about 0.4 mg / mL, about 0.6 mg / mL, about 0.8 mg / mL, or about 1 mg / mL. In some embodiments, the cationic drug in the cationic drug solution has a concentration of about 0.625 mg / mL.

[0112] In some embodiments, the concentration of the cationic drug in the cationic drug solution is about 0.5 to about 9 mg / mL, about 0.7 to about 7 mg / mL, about 0.9 to about 5 mg / mL, or about 2 to about 3 mg / mL. In some embodiments, the concentration of the cationic drug in the cationic drug solution is about 1 mg / mL, about 3 mg / mL, about 5 mg / mL, or about 7 mg / mL. In some embodiments, the concentration of the cationic drug in the cationic drug solution is about 2.5 mg / mL.

[0113] In some embodiments, the process also includes adding an additional surfactant to the loaded lipid nanoparticles (e.g., in addition to the cationic agent). In some embodiments, the additional surfactant is a PEG lipid, e.g., PEG-DMG. In some embodiments, the additional surfactant is provided with the cationic agent. In some embodiments, the additional surfactant is included with the cationic agent in the cationic agent solution. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.1 to about 50 mg / mL, about 1 to about 10 mg / mL, or about 1 to about 3 mg / mL.

[0114] In some embodiments, the additional surfactant and the cationic agent are provided separately.

[0115] In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.08 to about 0.9 mg / mL, about 0.1 to about 0.7 mg / mL, or about 0.3 to about 0.5 mg / mL. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.1 mg / mL, about 0.3 mg / mL, or about 0.4 mg / mL. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.36 mg / mL.

[0116] In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.05 to about 4.0 mg / mL, about 0.5 to about 3.0 mg / mL, or about 1.0 to about 2.0 mg / mL. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.5 mg / mL, about 1.5 mg / mL, or about 2.5 mg / mL. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 1.45 mg / mL.

[0117] For example, adding an additional surfactant to the composition can correspond to the PI buffer step of FIG. 8, FIG. 15, or FIG.

[0118] In some embodiments, the weight ratio of the cationic agent to payload is about 1:1 to about 4:1, about 1.25:1 to about 3.75:1, about 1.25:1, about 2.5:1, or about 3.75:1.

[0119] In some embodiments, the loaded lipid nanoparticle composition undergoes aging after loading and prior to neutralization with a controlled residence time, in some embodiments, the residence time is about 5 to about 120 seconds, about 10 to about 90 seconds, about 20 to about 70 seconds, about 30 to about 60 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0120] In some embodiments, the loaded lipid nanoparticle composition undergoes aging after neutralization and prior to addition of the cationic agent with a controlled residence time, in some embodiments, the residence time is about 1 to about 30 seconds, about 2 to about 20 seconds, about 5 to about 15 seconds, about 7 to about 12 seconds, or about 10 seconds.

[0121] In some embodiments, the process for preparing the 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; filtering the composition; concentrating the composition; exchanging the buffer of the composition; adding a cryoprotectant to the composition; and Adding an osmolality adjusting agent to the composition.

[0122] In some embodiments, the process of preparing the loaded lipid nanoparticle composition can further comprise one, two, three, four, five, six, seven or all of the steps listed above. Some steps can be repeated. The steps can be, but do not have to be, carried out in the order listed above. Each of the steps refers to an action related to the composition obtained from the step carried out before. For example, when the process comprises the step of adding one or more surfactants to the composition, the surfactants are added to the composition obtained from the previous step, and in this case, the previous step can be any of the steps listed above.

[0123] In some embodiments, the one or more further 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.

[0124] In some embodiments, the pH is adjusted to about pH 7.2.

[0125] In some embodiments, the pH is adjusted by adding a neutralization buffer. For example, the addition of a neutralization buffer may correspond to the neutralization buffer step of FIG. 8, FIG. 12, FIG. 15, or FIG. 16. 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 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% to about 22%, about 14% to about 20%, about 16% to about 18%, or about 17% w / v sucrose.

[0126] In some embodiments, the one or more further steps are adding surfactant to the composition.Surfactant can include but is not limited to PEG derivative (for example, PEG-DMG), lipid amine (for example, sterol amine and related substances), anionic protein (for example, bovine serum albumin), surfactant (for example, cationic surfactant, for example, dimethyldioctadecylammonium bromide), sugar or sugar derivative (for example, cyclodextrin), nucleic acid, polymer (for example, heparin, polyethylene glycol, and poloxamer), mucolytic agent (for example, acetylcysteine, mugwort, bromelain, papain, clover, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, tiopronin, gelsolin, thymosin β4, dornase alpha, neltenexin, and erdostein) and DNase (for example, rhDNase). The surfactant can be disposed within and / or on the surface of the nanoparticle (e.g., by coating, adsorption, covalent bonding, or other processes). For example, adding additional surfactant to the composition can correspond to the PI buffer step of FIG.

[0127] In some embodiments, the one or more further steps are adding an osmolality modifier to the composition. The osmolality modifier can be a salt or a sugar. In some embodiments, the osmolality modifier is a sugar. The sugar can be selected from, but not limited to, glucose, fructose, galactose, sucrose, lactose, maltose, and dextrose. In some embodiments, the osmolality modifier is a salt. The salt can 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 can 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 can be about 7 to about 8. The salt solution may further include a buffer, such as an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer, and 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.

[0128] In some embodiments, the osmotic agent is provided along with the cationic agent.

[0129] For example, adding an osmolality adjusting agent to the composition can correspond to the salt spiking steps of FIGS.

[0130] Cryoprotectants may be added to the loaded 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 included in the cryoprotectant solution includes 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 further embodiments, the buffer is an acetate buffer, e.g., 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% by weight, about 50% to about 85% by weight, about 60% to about 80% by weight, or about 70% by weight of sucrose.

[0131] In some embodiments, the cryoprotectant is provided along with the cationic agent.

[0132] For example, adding a cryoprotectant to the composition may correspond to the fill and finish steps of FIGS.

[0133] In some embodiments, the process includes diluting the composition with a dilution buffer. 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 further embodiments, 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 in the process of mixing the empty lipid nanoparticles with the nucleic acid solution.

[0134] In some embodiments, the process comprises any 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 exchanging steps can be performed using tangential flow filtration (TFF). Residual organic solvent can be removed by the filtering step.

[0135] For example, filtering the composition, concentrating the composition, and exchanging the buffer of the composition can correspond to the TFF filtration step of FIG. 8 or the TFF step of FIG. 15 or FIG.

[0136] In some embodiments, tangential flow filtration is performed after adding a cationic agent to the loaded lipid nanoparticle composition, hi some embodiments, tangential flow filtration is performed before adding a cationic agent to the loaded lipid nanoparticle composition.

[0137] In some embodiments, the filtration of the composition, the concentration of the composition, and the exchange of the buffer of the composition are performed after adding a cationic agent to the loaded lipid nanoparticle composition, hi some embodiments, the filtration of the composition, the concentration of the composition, and the exchange of the buffer of the composition are performed before adding a cationic agent to the loaded lipid nanoparticle composition.

[0138] In some embodiments, the composition is filtered after adding a cationic agent to the loaded lipid nanoparticle composition, hi some embodiments, the composition is filtered before adding a cationic agent to the loaded lipid nanoparticle composition.

[0139] In some embodiments, the buffer exchange may change the composition of the loaded lipid nanoparticle composition by increasing or decreasing the buffer concentration, changing the buffer composition, or changing the pH.

[0140] In some embodiments, the concentrating step may increase the concentration of loaded lipid nanoparticles in the composition.

[0141] In some embodiments, the process for preparing the loaded lipid nanoparticle composition further comprises at least the following steps: adjusting the pH of the composition to about pH 7 to about 8 (e.g., about pH 7.5) and adding an osmolality adjusting agent (e.g., an inorganic salt) to the composition.

[0142] In some embodiments, the process for preparing the loaded lipid nanoparticle composition further comprises at least the following steps: adjusting the pH of the composition to about pH 7 to about 8 (e.g., about pH 7.5), adding a surfactant to the composition, and adding an osmolality adjusting agent (e.g., an inorganic salt) to the composition.

[0143] The empty lipid nanoparticle composition and the nucleic acid solution are mixed to obtain a filled lipid nanoparticle composition. The mixing can 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 of, 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.

[0144] Generally speaking, the loaded lipid nanoparticle composition comprises nanoparticles having a larger average diameter than the starting empty particles. For example, the loaded nanoparticles 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. Additionally, the loaded lipid nanoparticle composition is characterized by a polydispersity index (PDI). For example, the polydispersity index (PDI) of the loaded lipid nanoparticle composition disclosed herein may be about 0.12 to about 0.25.

[0145] In some embodiments, the loaded lipid nanoparticle composition is contained 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 contained 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, e.g., sodium acetate. In some embodiments, the buffer is an acetate buffer and a 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.

[0146] In some embodiments, the storage solution comprises a cryoprotectant. In some embodiments, the cryoprotectant is one or more cryoprotectants, e.g., a polyol (e.g., a diol or triol, e.g., 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 sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 2 ... 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. 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 and sodium chloride.

[0147] In some embodiments, the concentration of loaded lipid nanoparticles 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.

[0148] 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.

[0149] In some embodiments, the process for preparing the loaded lipid nanoparticle composition further comprises: (i) adjusting the pH of the composition to about pH 7 to about 8; (ii) adding one or more surfactants to the composition; (iii) concentrating the composition; (iv) adding an inorganic salt to the composition; and (v) diluting the composition.

[0150] Some embodiments include a process for preparing a loaded lipid nanoparticle composition, the process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid solution containing PEG-lipids, with an aqueous buffer solution having a pH of about 4.5 or less to obtain a blank lipid nanoparticle composition; and (b) mixing the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition, wherein the payload is for delivery to an epithelial cell; (c) adjusting the pH of the composition; (d) adding a cationic drug to the composition along with an additional surfactant; (e) filtering the composition; (f) concentrating the composition; (g) exchanging the buffer of the composition; (h) adding an osmolality adjusting agent to the composition; and (i) adding a cryoprotectant to the composition.

[0151] Some embodiments include a process for preparing a loaded lipid nanoparticle composition, the process comprising: (a) (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid with an aqueous buffer solution having a pH of about 4.5 or less to obtain a blank lipid nanoparticle composition; (b) mixing the empty lipid nanoparticle composition with a payload to form a loaded lipid nanoparticle composition, wherein the payload is for delivery to an epithelial cell; (c) adjusting the pH of the composition; (d) adding additional surfactant; (e) filtering the composition; (f) concentrating the composition; (g) exchanging the buffer of the composition; (h) adding a cationic drug to the composition along with an osmolality adjusting agent; and (i) adding a cryoprotectant to the composition.

[0152] Some embodiments include a loaded lipid nanoparticle composition prepared by any of the processes described herein for preparing a loaded lipid nanoparticle composition.

[0153] Lipid Nanoparticle Compositions Also provided is an empty lipid nanoparticle composition, which contains some other components, such as water, organic solvent, buffer, cryoprotectant, pharmaceutical additive, or any combination thereof. The empty lipid nanoparticles are suitable for preparing loaded or filled lipid nanoparticle compositions for therapeutic or prophylactic use. The empty lipid nanoparticle composition can be provided in liquid form, where water and / or organic solvent are present in the composition, and the particles are suspended or otherwise present in a liquid medium. The empty lipid nanoparticle composition can also be provided in solid form, such as frozen or lyophilized form.

[0154] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid The empty lipid nanoparticle composition comprises an empty lipid nanoparticle comprising: (a) contains substantially no payload; (b) having a pH of about 3 to about 5; (c) characterized by a zeta potential of about 35 mV or greater.

[0155] The empty lipid nanoparticle compositions are substantially free of payload, e.g., substantially free of any therapeutic or prophylactic proteins or nucleic acids, and therefore are useful for preparing loaded or loaded lipid nanoparticles that contain payload.

[0156] The empty lipid nanoparticle composition may be characterized by having a relatively high zeta potential of about 35 mV or greater. In some embodiments, the empty lipid nanoparticle composition is characterized by a zeta potential of about 50 mV or greater, or about 100 mV or greater. In further 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 at a pH range of 3-6, at least about 33% of the maximum zeta potential achievable for the composition at a pH range of 3-6, at least about 50% of the maximum zeta potential achievable for the composition at a pH range of 3-6, at least about 66% of the maximum zeta potential achievable for the composition at a pH range of 3-6, or at least about 75% of the maximum zeta potential achievable for the composition at a pH range of 3-6.

[0157] The empty lipid nanoparticle composition can be characterized as having an acidic pH, for example, from about 3 to about 5. In some embodiments, the composition has a pH of about 3.5 to about 4.5. In further embodiments, the composition has a pH of about 4. In further embodiments, the composition has a pH of about 5.

[0158] The empty lipid nanoparticle compositions can 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.

[0159] The empty lipid nanoparticle composition can 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 compositions described herein can 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.

[0160] In some embodiments, the empty lipid nanoparticle composition has an empty lipid nanoparticle concentration 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.

[0161] In some embodiments, the empty lipid nanoparticle composition comprises a buffer. For example, the composition may have about 1 to about 100 mM buffer, about 1 to about 10 mM buffer, or about 5 mM buffer. A suitable buffer is any that can maintain an acidic pH at a relatively low ionic strength. Exemplary buffers include acetate buffer, citrate buffer, phosphate buffer, Tris buffer, or a combination thereof.

[0162] The empty lipid nanoparticle composition may further comprise a cryoprotectant, such as 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%, about 10 to about 30%, or about 20% w / v sucrose (or other cryoprotectant). In further embodiments, the empty lipid nanoparticle composition may comprise about 1 to about 15%, about 5 to about 10%, about 7 to about 8%, or about 7.5% w / v sucrose (or other cryoprotectant).

[0163] In some embodiments, the empty lipid nanoparticle composition of the present invention may further comprise an organic solvent. The organic solvent is usually miscible with water. Examples of organic solvents include alcohol, e.g., ethanol. In some embodiments, the organic solvent is present in an amount of about 25% by volume or less. In some embodiments, the empty lipid nanoparticle composition comprises about 25% by volume ethanol. 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.

[0164] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) PEG-lipid The empty lipid nanoparticle composition comprises about 1 to about 100 mg / mL of empty lipid nanoparticles comprising: (a) contains substantially no 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.

[0165] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a blank lipid nanoparticle composition comprising about 25 to about 75 mg / mL of blank lipid nanoparticles comprising PEG-lipids, The empty lipid nanoparticle composition comprises: (a) contains substantially no 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 comprising about 10 to about 30% w / v sucrose.

[0166] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a blank lipid nanoparticle composition comprising about 50 mg / mL of blank lipid nanoparticles comprising PEG-lipids; The empty lipid nanoparticle composition comprises: (a) contains substantially no 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 comprising about 20% w / v sucrose.

[0167] Some embodiments include a loaded lipid nanoparticle composition comprising loaded lipid nanoparticles and some other components, such as water, organic solvent, buffer, cryoprotectant, or any combination thereof. The loaded lipid nanoparticles are generally suitable for therapeutic or prophylactic use in patients. The loaded lipid nanoparticle composition can be provided in a liquid form, where water and / or organic solvent are present in the composition and the particles are suspended or otherwise present in a liquid medium. The loaded lipid nanoparticle composition can also be provided in a solid form, such as a frozen or lyophilized form.

[0168] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, (v) cationic drugs, and (vi) Payload and wherein the lipid nanoparticle composition has a pH of about 4.5 to about 8.

[0169] The loaded lipid nanoparticle composition may be prepared by loading an empty lipid nanoparticle composition as described herein. The loaded lipid nanoparticle composition may have a pH of about 5 to about 8. For example, in some embodiments of the loaded lipid nanoparticle composition obtained directly from loading, it may have a pH of about 5. In some embodiments, for example, when the loaded lipid nanoparticle composition is neutralized, it may have a pH of about 7 to about 8, e.g., about 7.5.

[0170] 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.

[0171] 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% to about 10%, about 1% to about 5%, or about 3% to about 4% w / v. In some embodiments, the cryoprotectant is sucrose.

[0172] In some embodiments, the loaded lipid nanoparticle composition further comprises an inorganic salt, such as any of the inorganic salts 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.

[0173] 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, about 30 mM to about 50 mM buffer, or about 40 mM buffer. In some embodiments, the buffer comprises acetate buffer or Tris buffer, or a combination thereof. In further embodiments, the buffer comprises acetate buffer and Tris buffer.

[0174] The loaded lipid nanoparticle composition can be further characterized according to the average diameter. The loaded lipid nanoparticles can have an average diameter larger than the starting empty particles. For example, the loaded nanoparticles can 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.

[0175] The loaded lipid nanoparticle compositions can be further characterized according to their polydispersity index (PDI). The loaded lipid nanoparticle compositions described herein can 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.

[0176] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, (v) cationic drugs, and (vi) Payload The filled lipid nanoparticle composition comprises a filled lipid nanoparticle composition having a pH of about 4.5 to about 8, the filled lipid nanoparticle composition comprising: (a) having a pH of about 7 to about 8; (b) 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.

[0177] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, (v) cationic drugs, and (vi) Payload The lipid nanoparticle composition has a pH of about 4.5 to about 8, and 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% 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.

[0178] Some embodiments comprise the following components: (i) ionic lipids, (ii) phospholipids, (iii) structured lipids; (iv) PEG-lipids, (v) cationic drugs, and (vi) Payload The lipid nanoparticle composition has a pH of about 4.5 to about 8, and 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.

[0179] In some embodiments, the payload is an mRNA, e.g., an mRNA encoding a protein expressed in airway epithelium, e.g., CFTR, short palate lung and nasal epithelium clone 1 (SPLUNC1), and alpha-1-antitrypsin (AAT).

[0180] In some embodiments, the empty or filled lipid nanoparticle composition comprises about 30 mol% to about 60, about 35 mol% to about 55 mol%, or about 40 mol% to about 50 mol% ionic lipid relative to total lipid.

[0181] 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.

[0182] 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.

[0183] 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 total lipid.

[0184] In some embodiments, the empty or filled lipid nanoparticle compositions each comprise, relative to total lipid: about 40 mol % to about 50 mol % of an ionic lipid; about 10 mol% to about 12 mol% of phospholipids, about 37 mol % to about 42 mol % of structural lipids, and About 0.25 mol% to about 0.75 mol% PEG-lipid.

[0185] In some embodiments, the lipid solution comprises, relative to total lipid, the following: Approximately 49 mol% ionic lipids, about 11 mol% to about 12 mol% of phospholipids, Approximately 39 mol% structural lipids, and Approximately 0.5 mol% PEG-lipid.

[0186] Any of the empty or filled lipid nanoparticle compositions provided herein can be prepared for storage or transportation. For example, the empty or filled lipid nanoparticle compositions 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 storage and / or transportation, for example, at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C.

[0187] Cationic Drugs The cationic agent can include any aqueous soluble molecule or substance that has a net positive charge and can adhere to the surface of the lipid nanoparticle core. Such agents can also be lipid soluble, but are also soluble in aqueous solutions. The cationic agent can be charged at physiological pH. Physiological pH is the pH level normally observed in the human body. Physiological pH can be about 7.30-7.45 or about 7.35-7.45. Physiological pH can be about 7.40. Generally speaking, the cationic agent is characterized by a net positive charge at physiological pH, since it contains one or more basic functional groups that are protonated at physiological pH in aqueous media. For example, the cationic agent can include one or more amine groups, such as primary, secondary, or tertiary amines, each having a pKa of 8.0 or more. The pKa can be greater than about 9.

[0188] In some embodiments, the cationic agent can be a cationic lipid, which is a water-soluble amphipathic molecule, where one part of the molecule is hydrophobic, e.g., includes a lipid moiety, and the other part of the molecule is hydrophilic and includes one or more functional groups that are usually charged at physiological pH. The hydrophobic part, including the lipid moiety, can serve to fix the cationic agent to the lipid nanoparticle core. The hydrophilic part can serve to increase the surface charge of the lipid nanoparticle core. For example, the cationic agent can have a solubility of more than about 1 mg / mL in alcohol. The solubility in the alcohol can be more than about 5 mg / mL. The solubility in the alcohol can be more than about 10 mg / mL. The solubility in the alcohol can be more than about 20 mg / mL in alcohol. The alcohol can have a C 1-6 It may be an alcohol, for example ethanol.

[0189] The lipid portion of the molecule can be, for example, a structured lipid, a fatty acid, or similar hydrocarbyl group.

[0190] The structured lipids may be selected from, but are not limited to, steroids, diterpenoids, triterpenoids, cholestane, ursolic acid, or derivatives thereof.

[0191] In some embodiments, the structured lipid is a steroid selected from, but not limited to, cholesterol or phystosterol.In some embodiments, the structured lipid is a cholesterol analog.In some embodiments, the structured lipid is sitosterol, campesterol, or stigmasterol.In some embodiments, the structured lipid is a sitosterol, campesterol, or stigmasterol analog.

[0192] The fatty acid is a fatty acid having 1 to 4 C 6-20 The fatty acids include a hydrocarbon chain. The fatty acids may be fully saturated or may contain 1 to 7 double bonds. The fatty acids may include 1 to 5 heteroatoms along or pendant to the backbone.

[0193] In some embodiments, the fatty acid has two C 10-18 In some embodiments, the fatty acid comprises two C 10-18 In some embodiments, the fatty acid comprises two C 16 In some embodiments, the fatty acid comprises two C 14 In some embodiments, the fatty acid comprises a saturated hydrocarbon chain. 10-18 In some embodiments, the fatty acid comprises two C 16-18 In some embodiments, the fatty acid comprises three C 8-18 Contains a saturated hydrocarbon chain.

[0194] The hydrocarbyl group is selected from 1 to 4 C 6-20 It consists of an alkyl, alkenyl, or alkynyl chain, or a 3-10 membered cycloalkyl, cycloalkenyl, or cycloalkynyl group.

[0195] In some embodiments, the hydrocarbyl chain is 8-10 In some embodiments, the hydrocarbyl chain is C8-10 It is alkenyl.

[0196] The hydrophilic portion may include 1 to 5 functional groups that are charged at physiological pH, i.e., 7.3 to 7.4. The hydrophilic groups may include basic functional groups that are protonated and positively charged at physiological pH. At least one of the basic functional groups has a pKa of 8 or higher.

[0197] In some embodiments, the hydrophilic moiety comprises an amine group. The amine group may comprise one to four primary, secondary, or tertiary amines and mixtures thereof. The primary, secondary, or tertiary amine may be part of a larger amine that contains a functional group selected from, but not limited to, -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N-. The amine may be contained in a 3-8 membered heteroalkyl or heteroaryl ring.

[0198] In some embodiments, the amine group comprises one or two terminal primary amines. In some embodiments, the amine group comprises one or two terminal primary amines and one internal secondary amine. In some embodiments, the amine group comprises one or two tertiary amines. In some embodiments, the tertiary amine is (CH3)2N-. In some embodiments, the amine group comprises one to two terminal (CH3)2N-.

[0199] The hydrophilic portion may comprise a phosphonium group, the counterion of which consists of a monovalent anion.

[0200] In some embodiments, three of the phosphonium substituents are isopropyl groups. In some embodiments, the counterion is a halo, hydrogen sulfate, nitrite, chloride, or hydrogen carbonate. In some embodiments, the counterion is a bromide.

[0201] In some embodiments, the cationic agent is a cationic lipid that is a sterol amine. A sterol amine has a sterol in its hydrophobic portion and an amine group in its hydrophilic portion. The sterol group is selected from, but not limited to, cholesterol, sitosterol, campesterol, stigmasterol, or derivatives thereof. The amine group may include 1-5 primary, secondary, tertiary amines, or mixtures thereof. At least one of the amines has a pKa of 8 or greater and is charged at physiological pH. The primary, secondary, or tertiary amine may be part of a larger amine that includes a functional group selected from, but not limited to, -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N-. The amine may be included in a 3-8 membered heteroalkyl or heteroaryl ring.

[0202] In some embodiments, the amine group of the sterol amine comprises one or two terminal primary amines. In some embodiments, the amine group comprises one or two terminal primary amines and one internal secondary amine. In some embodiments, the amine group comprises one or two tertiary amines. In some embodiments, the tertiary amine is (CH3)2N-. In some embodiments, the amine group comprises one to two terminal (CH3)2N-.

[0203] Sterol amines useful in the nanoparticles include those of formula (A1): ALB(A1) or a salt thereof, where A is an amine group, L is an optional linker, and B is a sterol.

[0204] In some embodiments, the amine group is an alkyl (e.g., C 1-14 Alkyl, C 1-12 Alkyl, C 1-10 alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), or C 1-6alkyl-(5-6 membered heteroaryl), where the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Alkyl-(5-6 membered heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or combinations thereof, where the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Each alkyl-(5- to 6-membered heteroaryl) is optionally 1-6 Alkyl, halo, OH, O(C 1-6 Alkyl), C 1-6 Alkyl-OH, NH2, NH(C 1-6 alkyl), N(C 1-6 alkyl) 2, 3-8 membered heterocycloalkyl (optionally containing 1-5 primary, secondary, or tertiary amines, or a combination thereof 1-14 In some embodiments, the linker is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, or -SS-CH2-CH2-C(O)N-. In some embodiments, the sterol group is cholesterol, sitosterol, campesterol, stigmasterol, or a derivative thereof.

[0205] In some embodiments, the sterol amine has the formula A2a: [ka] or a salt thereof, wherein ---- is a single or double bond, R 1 is C1-14 Alkyl or C 1-14 alkenyl, L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] Based on Y 1 is C 1-10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), or C 1-6 alkyl-(5-6 membered heteroaryl); wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Each alkyl-(5- to 6-membered heteroaryl) is optionally 1-6 Alkyl, halo, OH, O(C 1-6 Alkyl), C 1-6 Alkyl-OH, NH2, NH(C 1-6 alkyl), N(C 1-6 alkyl) 2, 3-8 membered heterocycloalkyl (optionally containing 1-5 primary, secondary, or tertiary amines, or a combination thereof 1-14 substituted with one, two, three, or four substituents selected from: NH(3-8 membered heterocycloalkyl), NH(5-6 membered heteroaryl), n=1 or 2.

[0206] In some embodiments, the sterol amine has the formula A3a: [ka] or a salt thereof, wherein ---- is a single or double bond, R 2 is H or C 1-6 is alkyl, L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] Based on Y 1 is C 1-10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), or C 1-6 alkyl-(5-6 membered heteroaryl); wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Each alkyl-(5- to 6-membered heteroaryl) is optionally 1-6 Alkyl, halo, OH, O(C 1-6 Alkyl), C 1-6Alkyl-OH, NH2, NH(C 1-6 alkyl), N(C 1-6 alkyl) 2, 3-8 membered heterocycloalkyl (optionally containing 1-5 primary, secondary, or tertiary amines, or a combination thereof 1-14 substituted with one, two, three, or four substituents selected from: NH(3-8 membered heterocycloalkyl), NH(5-6 membered heteroaryl), n=1 or 2.

[0207] In some embodiments, the sterol amine has formula A4: [ka] or a salt thereof, wherein Z 1 is OH or C 3-6 is alkyl, L is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, or -SS-CH2-CH2-C(O)N-; Y 1 is C 1-10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), or C 1-6 alkyl-(5-6 membered heteroaryl); wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C 1-6 Alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1-6 Alkyl-(3-8 membered heterocycloalkyl), and C1-6 Each alkyl-(5- to 6-membered heteroaryl) is optionally 1-6 Alkyl, halo, OH, O(C 1-6 Alkyl), C 1-6 Alkyl-OH, NH2, NH(C 1-6 alkyl), N(C 1-6 alkyl) 2, 3-8 membered heterocycloalkyl (optionally containing 1-5 primary, secondary, or tertiary amines, or a combination thereof 1-14 substituted with one, two, three, or four substituents selected from: NH(3-8 membered heterocycloalkyl), NH(5-6 membered heteroaryl), n=1 or 2.

[0208] In some embodiments, the sterol amine has formula A5: [ka] or a salt thereof, wherein Z 2 is OH or isopropyl, L 3 is -CH2-NH-C(O)-, -C(O)NH-, or -C(O)O-.

[0209] In some embodiments, Y 1 teeth, [ka] (28)N(CH3)2; [ka] is selected from.

[0210] In some embodiments, the sterol amine is [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] or a salt thereof.

[0211] In some embodiments, the sterol amine is SA3: [ka] or a salt thereof, which is also referred to as GL-67. SA3 or GL-67 may be prepared according to processes known in the art or purchased from a commercial supplier such as Avanti® Polar Lipids, Inc. (SKU 890893).

[0212] In some embodiments, the cationic lipid is a modified amino acid, such as modified arginine, in which an amino acid residue with an amine-containing side chain is added to a hydrophobic group, such as sterol (e.g., cholesterol or its derivative), fatty acid, or similar hydrocarbyl group. At least one amine of the modified amino acid moiety has a pKa of 8.0 or higher. At least one amine of the modified amino acid moiety is positively charged at physiological pH. The amino acid residue can include, but is not limited to, arginine, histidine, lysine, tryptophan, ornithine, and 5-hydroxylysine. The amino acid is linked to the hydrophobic group via a linker.

[0213] In some embodiments, the modified amino acid is a modified arginine.

[0214] In some embodiments, the cationic agent is a non-lipid cationic agent. Examples of non-lipid cationic agents include, for example, benzalkonium chloride, cetylpyridinium chloride, L-lysine monohydrate, or tromethamine.

[0215] Ionic Lipids As used herein, the term "ionic lipid" has its usual meaning in the art and may refer to a lipid that contains one or more charged moieties. In some embodiments, an ionic lipid may be positively or negatively charged. For example, an ionic lipid may be positively charged at low pH, in which case it may be referred to as a "cationic lipid". In certain embodiments, an ionic lipid molecule may contain an amine group and may be referred to as an ionic amino lipid. As used herein, a "charged moiety" is a chemical moiety that carries a formal electronic charge, such as monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. The 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 the charged moiety may in some cases vary depending on 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 the molecule may be selected as desired.

[0216] In some embodiments, the nanoparticles described herein comprise about 30 mol% to about 60 mol% ionic lipid. In some embodiments, the nanoparticles described herein comprise about 35 mol% to about 55 mol% ionic lipid. In some embodiments, the nanoparticles comprise about 40 mol% to about 50 mol% ionic lipid. In some embodiments, the nanoparticles comprise about 45 mol% to about 50 mol% ionic lipid.

[0217] 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.

[0218] In some embodiments, the ionic 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 each 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.

[0219] In some embodiments, the ionic 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 each H, 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.

[0220] In some embodiments, the ionic 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 each H, 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.

[0221] In some embodiments, the ionic 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 each H, 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.

[0222] In some embodiments, the ionic 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 each H, 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.

[0223] In some embodiments, the ionic lipid is [ka] or an N-oxide or salt thereof.

[0224] In some embodiments, the ionic lipid comprises the compound: [ka] or an N-oxide or salt thereof.

[0225] In some embodiments, the ionic lipid comprises the compound: [ka] or an N-oxide or salt thereof.

[0226] In some embodiments, the ionic lipid comprises the compound: [ka] or an N-oxide or salt thereof.

[0227] In some embodiments, the ionic lipid comprises the compound: [ka] or an N-oxide or salt thereof.

[0228] In some embodiments, the ionic 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 each 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, I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0229] In some embodiments, the ionic 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 , Raβ , R aγ , and R aδ are each 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, I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0230] In some embodiments, the ionic 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 each H, R 2and 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.

[0231] In some embodiments, the ionic 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 each H, 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.

[0232] In some embodiments, the ionic 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 each H, 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.

[0233] In some embodiments, the ionic 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 each 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, I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0234] In some embodiments, R1 teeth, [ka] and [ka] indicates the attachment point, R aβ , R aγ , and R aδ are each H, 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.

[0235] In some embodiments, the ionizable lipid of formula (I) is [ka] or an N-oxide or salt thereof.

[0236] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 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 each 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 each 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 1-14Alkyl 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.

[0237] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 teeth, [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγ and R aδ are each 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 each 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.

[0238] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 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.

[0239] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 teeth, [ka] and R' b teeth, [ka] and [ka] indicates the attachment point, R aγis 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.

[0240] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 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.

[0241] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R'a is R' 分岐 or R' 環状 and R' 分岐 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.

[0242] In some embodiments, m and l are each independently selected from 4, 5, and 6. In some embodiments, m and l are each 5.

[0243] In some embodiments, each R' is independently C 1-12 In some embodiments, each R' is independently C 2-5 It is an alkyl.

[0244] In some embodiments, R'b teeth, [ka] and R 2 and R 3 are each independently 1-14 It is an alkyl.

[0245] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each independently 6-10 It is an alkyl.

[0246] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each C alkyl.

[0247] In some embodiments, R' 分岐 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.

[0248] In some embodiments, R' 分岐 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 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 is alkyl, R 2 and R 3 are each C alkyl.

[0249] In some embodiments, R' 分岐 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are respectively, C 1-12 It is an alkyl.

[0250] In some embodiments, R' 分岐 teeth, [ka] and R' b teeth, [ka] and R aγ and Rbγ are respectively, C 2-6 It is an alkyl.

[0251] In some embodiments, m and l are each independently selected from 4, 5, and 6, and each R' is independently selected from C 1-12 In some embodiments, m and l are each 5 and each R' is independently C 2-5 It is an alkyl.

[0252] In some embodiments, R' 分岐 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 respectively, C 1-12 It is an alkyl.

[0253] In some embodiments, R' 分岐 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 respectively, C 2-6 It is an alkyl.

[0254] In some embodiments, R' 分岐 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.

[0255] In some embodiments, R' 分岐 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 C alkyl.

[0256] In some embodiments, R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), and n2 is 2.

[0257] In some embodiments, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0258] In some embodiments, R' 分岐 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 respectively, C 1-12 is alkyl, R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), and n2 is 2.

[0259] In some embodiments, R' 分岐 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 respectively, C 2-6 is alkyl, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0260] In some embodiments, R' 分岐 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.

[0261] In some embodiments, R' 分岐 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, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0262] 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.

[0263] In some embodiments, R' 分岐 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 respectively, C 1-12 is alkyl, R 4 is -(CH2) n OH and n is 2, 3, or 4.

[0264] In some embodiments, R' 分岐 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 respectively, C 2-6 is alkyl, R 4 is -(CH2) n OH and n is 2.

[0265] In some embodiments, the ionic lipid has formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐 or R' 環状 and R' 分岐 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.

[0266] In some embodiments, m and l are each 5 and n is 2, 3, or 4.

[0267] In some embodiments, R' is C 2-5 is alkyl, R aγ is C 2-6 is alkyl, R 2 and R 3 are respectively, C 6-10 It is an alkyl.

[0268] 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 respectively, C 6-10 It is an alkyl.

[0269] In some embodiments, the ionic 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.

[0270] In some embodiments, the ionic 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 C2-5 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.

[0271] In some embodiments, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0272] In some embodiments, R 4 is -(CH2)2OH.

[0273] In some embodiments, the ionic 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” is selected. 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'.

[0274] 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 3are each -C(O)-.

[0275] In some embodiments, the compound of formula (III) is [ka] It is.

[0276] In some embodiments, the compound of formula (I) is [ka] It is.

[0277] In some embodiments, the ionic lipid is [ka] [ka] It is.

[0278] mRNA-lipid adducts It has been found that certain ionic lipids are prone to form lipid-polynucleotide adducts.In particular, ionic lipids that contain tertiary amine groups can decompose into secondary amines and / or reactive aldehyde species that can interact with polynucleotides (such as mRNA), forming ionic lipid-polynucleotide adduct impurities, which can be detected by reversed-phase ion-pair chromatography (RP-IP HPLC).For example, the oxidation of the tertiary amines can form N-oxides, which can undergo acid / base catalyzed hydrolysis at the amines to produce aldehydes and secondary amines that can form adducts with mRNA.Therefore, in some embodiments, the ionic lipid-polynucleotide adduct impurities are aldehyde-mRNA adduct impurities.

[0279] It has also been found that such adducts may interfere with the translation of mRNA and may affect the activity of mRNA products formulated in lipid nanoparticles (LNPs).Therefore, it may be advantageous to prepare and use LNP compositions with reduced content of ionic lipid-polynucleotide adduct impurities, for example, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the mRNA in the form of ionic lipid-polynucleotide adduct impurities that can be measured by RP-IP HPLC.Thus, in some embodiments, LNP compositions are provided with less than about 10%, less than about 5%, or less than about 1%, including less than 10%, less than 5%, or less than 1% of the mRNA in the form of ionic lipid-polynucleotide adduct impurities that can be measured by RP-IP HPLC.

[0280] In some embodiments, the amount of lipid aldehydes 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 compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of transition metals, 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 compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of aldehyde compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some embodiments, the amount of ketone compounds 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 compounds in the composition is less than about 50 ppm, including less than 50 ppm.

[0281] In some embodiments, the composition is stable against the formation of ionic lipid-polynucleotide adduct impurities. In some embodiments, the amount of ionic 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 ionic 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 ionic lipid-polynucleotide adduct impurities in the composition increases at an average rate of less than about 0.5% per day, when stored at refrigerated temperature, optionally the refrigerated temperature is about 5° C.

[0282] Lipid vehicle (e.g., LNP) compositions with reduced content of impurities of ionic lipid-polynucleotide adducts can be prepared by a method of inhibiting the formation of either or both of N-oxides and aldehydes. Such methods can include treating a composition containing an ionic lipid containing a tertiary amine group to inhibit the formation of either or both of N-oxides and aldehydes, for example, 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 contained in the composition. Such methods can include one or more of treating the ionic lipid with a scavenger, treating the ionic lipid with a reducing agent, treating the ionic lipid with a chelating agent, treating the polynucleotide with a reducing agent, and treating the polynucleotide with a chelating agent before mixing the ionic lipid with the polynucleotide.

[0283] According to any of the above, the scavenger, reductive treatment agent, and / or reducing agent may be an agent that reacts with aldehydes, ketones, anhydrides, and / or diene compounds.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 reductive treating agent may include a boron compound (e.g., sodium borohydride and / or bis(pinacolato)diboron). The reductive treating agent may include a boron compound, e.g., 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, e.g., silica immobilized diphenylphosphine (Si-DPP), agarose immobilized thiol (Ag-thiol), silica immobilized cysteine ​​(Si-cysteine), silica immobilized thiol (Si-thiol), or a combination 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 a combination thereof.

[0284] According to any of the above, the pH may be about pH 7 to about pH 9, or may be adjusted thereto.

[0285] 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, and may be about 20 mM to about 150 mM TRIS, or may be adjusted thereto.

[0286] According to any of the above, the temperature of the composition may be, or may be adjusted to, below 25°C.

[0287] The composition may also include a free reducing agent or antioxidant.

[0288] PEGylated lipids The PEG lipid component of the lipid nanoparticle composition can include one or more molecules that include polyethylene glycol, such as 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.

[0289] In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.1 mol% and about 10 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.1 mol% and about 5 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.1 mol% and about 3 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.1 mol% and about 2 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.1 mol% and about 1 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.25 mol% and about 0.75 mol% PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise between about 0.5 mol% PEG-lipid.

[0290] 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 PEG-c-DOMG, PEG-DMG (e.g., PEG-DMG 2000 or DMG-PEG 2000), PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0291] 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-DMG 2000 (or DMG-PEG 2000), where 2000 represents the average molecular weight. A representative PEG-DMG structure is: [ka]

[0292] In one embodiment, the PEG lipid may be a PEGylated lipid, such as those described in International Publication No. WO2012 / 099755, the contents of which are incorporated herein by reference in their entirety. 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. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") 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.

[0293] In certain embodiments, the PEG lipid is a compound of formula (VII). Provided herein are compounds 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 from 1 to 100, including the endpoints; L 1 is an arbitrarily substituted C 1-10 alkylene, 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 )-, D is a moiety obtained by click chemistry or a moiety 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 1-6 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 )-, 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 are independently selected from 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(RN )-, -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, 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0298] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0299] In certain embodiments, the compound of formula (VII) has the following formula: [ka] In the formula, r is one of 1 to 100, or a salt thereof.

[0300] 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.

[0301] 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.

[0302] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0303] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0304] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0305] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0306] In certain embodiments, the PEG lipid is a PEGylated fatty acid. In certain embodiments, the PEG lipid is a compound of formula (VIII). Provided herein is a compound of 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 from 1 to 100, including the endpoints; R 5 is an arbitrarily substituted C 10-40Alkyl, 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-, RN Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0307] In certain embodiments, the compound of formula (VIII) has the formula (VIII-OH): [ka] or a salt thereof.

[0308] 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.

[0309] In yet another embodiment, the compound of formula (VIII) has the formula: [ka] or a salt thereof.

[0310] In some embodiments, the compound of formula (VIII) is [ka] It is.

[0311] In certain embodiments, the PEG lipid has the following formula: [ka] In some embodiments, r is 45, or a salt thereof.

[0312] Further suitable PEG lipids are described in WO2017 / 099823, which is incorporated by reference in its entirety.

[0313] 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, such as one or more (poly)unsaturated lipids. The phospholipids may be organized 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).

[0314] In some embodiments, the lipid nanoparticle compositions described herein may comprise from about 1 mol% to about 20 mol% phospholipid. In some embodiments, the lipid nanoparticle compositions described herein may comprise from about 5 mol% to about 15 mol% phospholipid. In some embodiments, the nanoparticle compositions comprise from about 8 mol% to about 13 mol% phospholipid. In some embodiments, the nanoparticle compositions comprise from about 10 mol% to about 12 mol% phospholipid.

[0315] Suitable phospholipids include 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 ... 1,2-Diundecanoyl-sn-glycero-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 (ME16.0PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME16:0PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME16:0PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L -serine (sodium salt) (4ME16:0PS), 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.

[0316] 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 (4ME16:0PE) [ka] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (4ME16:0PC) [ka] 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(sodium salt) (4ME16:0PG), [ka] 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME16:0PS) [ka] or a mixture thereof.

[0317] Further examples of suitable phospholipids include, but are not limited to, the following: [ka] [ka]

[0318] In certain embodiments, the phospholipid has formula (IX): [ka] or a salt thereof, wherein Each R 1are independently H or optionally substituted alkyl, or optionally, two R 1 together with the intervening atoms form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 together with the intervening atoms 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] 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 )-, 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 are independently selected from 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 NC(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] wherein R 2 Each instance of is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0319] In certain embodiments, a suitable 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 together with the intervening atoms form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 together with the intervening atoms 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 )-, R2 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 are independently selected from 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(RN )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] wherein R 2 Each instance of is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0320] In some embodiments, the compound has the formula: [ka] In the formula, R 2 Each instance of is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0321] 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.

[0322] In certain embodiments, the compound of formula (IX) is: [ka] [ka] or a salt thereof.

[0323] In certain embodiments, the compound of formula (IX) has the formula (IX-a): [ka] or a salt thereof.

[0324] In certain embodiments, suitable phospholipids include modified cores.In certain embodiments, the phospholipids having modified cores as 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.

[0325] In certain embodiments, the compound of formula (IX-b-4) has the following formula: [ka] or a salt thereof.

[0326] In certain embodiments, the compound of formula (IX) is: [ka] or a salt thereof.

[0327] 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) is represented by formula (IX-b): [ka] or a salt thereof.

[0328] 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.

[0329] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-2): [ka] or a salt thereof.

[0330] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-3): [ka] or a salt thereof.

[0331] In certain embodiments, the compound of formula (Ib) has the formula (Ib-4): [ka] or a salt thereof.

[0332] In certain embodiments, the compound of formula (IX-b) is: [ka] or a salt thereof.

[0333] In certain embodiments, the suitable phospholipid comprises a modified tail. In certain embodiments, the phospholipid is DSPC or an analog thereof with a modified tail. As described herein, the "modified tail" can 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 example of R 2 Each example of 1-30 alkyl, where R 2 one or more methylene units are independently selected from 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(RN )-, -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- is replaced by

[0334] 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 from 0 to 30, inclusive; Each instance of G is 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 )-, -NRN 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 possibility represents a separate embodiment.

[0335] 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.

[0336] In certain embodiments, the compound of formula (IX-c) has the formula (IX-c-2): [ka] or a salt thereof.

[0337] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0338] In certain embodiments, the compound of formula (IX-c) is: [ka] or a salt thereof.

[0339] In certain embodiments, the compound of formula (IX-c) has the formula (Ic-3): [ka] or a salt thereof.

[0340] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0341] In certain embodiments, the compound of formula (IX-c) is: [ka] or a salt thereof.

[0342] In certain embodiments, suitable phospholipids include 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) is the following formula: [ka] or a salt thereof.

[0343] In certain embodiments, the compound of formula (IX) is: [ka] [ka] or a salt thereof.

[0344] In certain embodiments, alternative lipids are used in place of phospholipids. Non-limiting examples of such alternative lipids include the following: [ka] Examples include:

[0345] structured lipids The lipid nanoparticle composition may include one or more structured lipids. The incorporation of structured lipids into the lipid nanoparticles 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]

[0346] 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 composition comprises about 30 mol% to about 50 mol% structured lipid. In some embodiments, the lipid nanoparticle composition comprises about 35 mol% to about 45 mol% structured lipid. In some embodiments, the lipid nanoparticle composition comprises about 37 mol% to about 42 mol% structured lipid. In some embodiments, the lipid nanoparticle composition comprises 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 a compound having the following structure: [ka]

[0347] Therapeutic and prophylactic drugs 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. Typically, the therapeutic agent delivered by the composition is a nucleic acid, but non-nucleic acid agents, such as small molecules, chemotherapeutic agents, peptides, polypeptides, and other biomolecules, are also payloads encompassed by the present disclosure. The 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).

[0348] 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).

[0349] In one embodiment, the therapeutic agent is a DNA therapeutic agent.The DNA molecule can be double-stranded DNA, 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 or linear DNA molecule.

[0350] 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 and expressing a transcript. 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.

[0351] The DNA therapeutics described herein, for example, DNA vectors, can include a variety of different features. The DNA therapeutics described herein, for example, DNA vectors, can include non-coding DNA sequences. For example, the DNA sequence can include at least one regulatory element of a gene, for example, 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 can have a non-coding DNA sequence operably linked to a transcriptionally active gene. In other embodiments, the DNA sequence described herein can have a non-coding DNA sequence that is not linked to a gene, i.e., the non-coding DNA does not control the gene of the DNA sequence.

[0352] In one embodiment, the therapeutic agent is an RNA therapeutic agent.The RNA molecule can be single-stranded RNA, double-stranded RNA (dsRNA) or partially double-stranded RNA molecule, i.e., the molecule has double-stranded and single-stranded parts.The RNA molecule can be circular or linear RNA molecule.

[0353] The RNA therapeutic agent can be an RNA therapeutic agent that can introduce a gene into cells, for example, encodes 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 generated in vitro.

[0354] Non-limiting examples of RNA therapeutics include messenger RNA (mRNA) (e.g., encoding a protein of interest), modified mRNA (mmRNA), mRNA introducing 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.

[0355] The mRNA may be naturally occurring or non-naturally occurring. The mRNA may contain one or more modified nucleic acid bases, nucleosides, or nucleotides, as described below, in which case it may be referred to as "modified mRNA" or "mmRNA". As used herein, a "nucleoside" is defined as a compound that includes a combination of a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group.

[0356] 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, substitutions, modifications, or other non-natural occurrences of the standard species. In certain embodiments, all of a particular nucleic acid base type may be modified.

[0357] 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.

[0358] The 5' cap structure or cap species is a compound that contains two nucleoside moieties linked by a linker and can 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 can contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can contain a guanine nucleotide and a guanine (G) nucleotide methylated at the 7th position linked at their 5' positions by a triphosphate bond, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. The cap species can 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'GppppG, m27,O3'GppppG, and m27,O2'GppppG.

[0359] The mRNA may alternatively or additionally comprise a chain-terminating nucleoside. For example, the chain-terminating nucleoside may include a nucleoside 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 mRNA may be stabilized by incorporating a chain-terminating nucleotide into the mRNA, for example, at the 3' end, as described, for example, in International Patent Publication WO2013 / 103659.

[0360] The mRNA may alternatively or additionally comprise a stem loop, e.g., 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 of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0361] The mRNA may alternatively or additionally comprise a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or mainly 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 export, translation, and / or stability of the mRNA.

[0362] The mRNA may alternatively, or in addition, contain a microRNA binding site.

[0363] In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region, with an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows for 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 typically used to enhance 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 IRES of encephalomyocarditis virus.

[0364] 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, improved translation, and / or lack of substantial induction of an innate immune response in 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 reduce immunogenicity.

[0365] 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 can be degraded less in the cell into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0366] 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.

[0367] 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 ... 5-methyl-2-thiocytidine (s2C), 2-thio-5-methyl-2-thiocytidine, 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.

[0368] 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.

[0369] 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*), 7-deaza-guanosine, queuosine (Q), epoxywyosine (Q), cyclohexyl ... Queuosine (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-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine guanosine, 1-methyl-guanosine (m1G), N2-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 2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 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.

[0370] 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).

[0371] 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 includes a combination of one or more of the modified nucleobases described above (e.g., a combination of two, three, or four of the modified nucleobases described above). 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 uracil contained in the mRNA. In some embodiments, N1-methylpseudouridine (m1Ψ) represents 100% of the uracil contained in the mRNA.

[0372] 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).

[0373] 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 modified nucleobases described above (e.g., combinations of two, three, or four of the modified nucleobases described above).

[0374] 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).

[0375] 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 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).

[0376] 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-thiouridine (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-methyl-uridine 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).

[0377] In certain embodiments, the mRNA of the present disclosure is uniformly modified with respect to a particular modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA may be uniformly modified with N1-methylpseudouridine (m1Ψ) or 5-methyl-cytidine (m5C), meaning that all uridine or all cytosine nucleosides contained in the mRNA sequence are replaced with N1-methylpseudouridine (m1Ψ) or 5-methyl-cytidine (m5C). Similarly, the mRNA of the present disclosure may be uniformly modified with respect to any type of nucleoside residue present in the sequence by substitution with modified residues such as those described above.

[0378] 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 comprise one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[0379] 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 Publication Nos. WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0380] 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.

[0381] When a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of its A, U, G, or C nucleotides or nucleosides being 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, 75% of the cytosines are CTP, 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 nucleotide sites found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unmodified.

[0382] The mRNA or regions thereof of the present disclosure can be codon-optimized. Codon optimization methods are known in the art and can be useful for various purposes, namely, to match codon frequency in host organisms and ensure proper folding, to bias GC content to enhance mRNA stability or reduce secondary structure, to minimize tandem repeat codons or runs of bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein transport sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of encoded proteins, to add, remove or replace protein domains, to insert or remove restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust translation rate and correctly fold various domains of proteins, or to reduce or remove problematic secondary structures in 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 sequence of the mRNA is optimized using an optimization algorithm, for example, to optimize expression in mammalian cells or to increase mRNA stability.

[0383] 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.

[0384] 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 used. 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 No. 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.

[0385] Non-natural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications can be present in the internucleoside linkage, the purine or pyrimidine base, or the sugar. In certain embodiments, the modifications can be introduced at the end of the polynucleotide chain or elsewhere in the polynucleotide chain by chemical synthesis or by 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).

[0386] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or regions thereof to 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).

[0387] In some embodiments, the payload therapeutic is a therapeutic 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 mRNAs that introduce microRNA binding site(s) (miR binding sites), microRNAs (miRNAs), antagomirs, small interfering RNAs (siRNAs) (including shortmers and dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs), and CRISPR / Cas9 technology.

[0388] In one embodiment, the therapeutic agent is a peptide therapeutic agent. In one embodiment, the therapeutic agent is a polypeptide therapeutic agent.

[0389] In some embodiments, the peptide or polypeptide is naturally derived, 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 less than 3, less than 5, less than 10, less than 15, less than 20, or less than 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0390] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising any of the lipid nanoparticle compositions described herein together with one or more pharma- ceutically acceptable excipients.

[0391] The pharmaceutical composition may optionally contain one or more additional active substances, e.g., therapeutic and / or prophylactic substances. The pharmaceutical composition of the present disclosure may be sterile and / or pyrogen-free. A general guide to the formulation and / or manufacture of pharmaceutical products can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005, incorporated herein by reference in its entirety. In some embodiments, the compositions are administered to a human, human patient or 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.

[0392] 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.

[0393] Pharmaceutical compositions according to the present disclosure may 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 that would be administered to a subject and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0394] The relative amounts of the active ingredient, pharma- ceutically acceptable excipients, and / or any additional components 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 on the route by which the composition is administered.

[0395] 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 understand that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals.

[0396] As used herein, pharma- ceutically acceptable additives include, but are not limited to, any solvent, dispersion medium, or other liquid medium, dispersion or suspension aid, diluent, granulating and / or dispersing agent, surfactant, isotonicity agent, thickening or emulsifying agent, preservative, binder, lubricant or oil, coloring agent, sweetener or flavoring agent, stabilizer, antioxidant, antibacterial or antifungal agent, osmolality adjusting agent, pH adjusting agent, buffer, chelating agent, cryoprotectant, and / or bulking agent, as appropriate for the particular dosage form desired. Various additives for formulating pharmaceutical compositions and techniques for preparing said 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).

[0397] 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.

[0398] The pharmaceutical compositions may be administered in an amount effective to produce a desired biological effect, e.g., a therapeutic or prophylactic effect, e.g., in some embodiments, an effect due to expression of a normal gene product, to replenish or replace a defective protein, or to reduce undesirable protein expression, as measured by the alleviation of one or more symptoms. The formulations may be administered in an amount effective to deliver the LNP.

[0399] Pharmaceutical compositions can be prepared in a variety of forms suitable for different 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.

[0400] 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 oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, oral compositions can 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.

[0401] 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 also be as sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, 1,3-butanediol solutions. 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 non-irritating fixed oils can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0402] Injectable formulations can be sterilized prior to use, 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.

[0403] The pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration. Such formulations may include dry particles containing the active ingredient. Such compositions may be in the form of a dry powder for administration using a device that includes a dry powder reservoir that can direct a stream of propellant to disperse the powder, and / or using a device that includes a self-propelling solvent / powder dispensing container, e.g., a sealed container that includes the active ingredient dissolved and / or suspended in a low-boiling propellant. Dry powder compositions may include a solid fine powder diluent, e.g., sugar, and may be provided in a unit dose form.

[0404] Low boiling point propellants generally include liquid propellants having a boiling point below about 65° F. at atmospheric pressure. Generally, the propellant may comprise 50% to 99.9% (wt / wt) of the composition, and the active ingredient may comprise 0.1% to 20% (wt / wt) of the composition. The propellant may further comprise additional ingredients, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size of the same order as the particles containing the active ingredient).

[0405] 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 optionally sterile aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any nebulizer and / or atomizer device. Such formulations may further include one or more additional ingredients, 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 a mean diameter ranging from about 1 nm to about 200 nm.

[0406] 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 containing the active ingredient, having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered by rapid inhalation through the nasal passages from a container of powder held close to the nostrils. Formulations suitable for nasal administration may, for example, contain as little as about 0.1% (wt / wt) to as much as 100% (wt / wt) of the active ingredient, and may contain one or more of the additional ingredients described herein.

[0407] method The loaded lipid nanoparticles described herein can be used in a method of delivering a payload to a cell, the method comprising contacting the cell with a loaded lipid nanoparticle composition described herein. The cell can be part of an in vitro or ex vivo sample. The cell can also be present in a patient. In some embodiments, the cell is an airway epithelial cell. The delivery of the payload can be by any of the means described herein, including administration of the loaded lipid nanoparticle composition to a patient by pulmonary delivery.

[0408] The loaded lipid nanoparticles described herein are also useful for treating or preventing diseases. In particular, such compositions may be useful for treating diseases characterized by missing or abnormal protein or polypeptide activity. In some embodiments, loaded lipid nanoparticle compositions described herein loaded with a payload such as an mRNA encoding a missing or abnormal polypeptide may be administered or delivered to cells. Subsequent translation of the mRNA produces a polypeptide, which may reduce or eliminate problems caused by the missing or abnormal activity caused by the polypeptide. Since translation may occur rapidly, the methods and compositions may 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 alter the transcription rate of a given species, thereby affecting gene expression.

[0409] 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.

[0410] In some embodiments, the LNPs have improved properties upon administration to cells, e.g., in vitro and in vivo, e.g., improved delivery of a payload to epithelial cells, e.g., as measured by cellular accumulation of the LNPs, expression of a desired protein, and / or mRNA expression.

[0411] The present disclosure provides methods that include administering one or more therapeutic and / or prophylactic agents, such as lipid nanoparticle compositions loaded with nucleic acids, as well as pharmaceutical compositions comprising the same. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for the prevention, treatment, diagnosis, or imaging of a disease, disorder, and / or condition, and / or 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 specific composition, the method of administration, and the like. Compositions according to the present disclosure may be formulated in dosage unit form 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 specific therapeutically effective dose, prophylactically effective dose, or other appropriate dose level (e.g., for imaging) for any particular patient will depend on a variety of factors. Such factors include 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 condition, 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 conjunction or concomitantly with the particular pharmaceutical composition used, and similar factors well known in the medical arts.

[0412] Combination therapy Lipid nanoparticle compositions loaded with one or more payload therapeutics and / or prophylactic agents, e.g., nucleic acids, may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" does not mean 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 compositions may be administered simultaneously with, prior to, or after one or more other desired therapeutics 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 the delivery of compositions in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution in the body, or imaging, diagnostic, or prophylactic compositions.

[0413] It will be further understood that therapeutic, preventive, diagnostic, or imaging agents used in combination may be administered together in a single composition or separately in different compositions. In general, it is expected that agents used in combination will be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination may be lower than the levels at which they are used 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 used may achieve a desired effect for the same disorder (e.g., a composition useful for treating cancer may be administered simultaneously with a chemotherapeutic agent) or they may achieve a different effect (e.g., control of any side effects, such as infusion-related reactions).

[0414] 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 modulator (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, a method of treating a subject in need of treatment or a 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 before administering the lipid nanoparticle composition.

[0415] Kits and Apparatus The present disclosure provides kits for convenient and / or effective use of the lipid nanoparticle compositions of the present disclosure. Typically, the kits contain sufficient amounts and / or numbers of components to enable a user to perform multiple treatments of a subject(s) and / or to enable a user to perform multiple experiments.

[0416] In one aspect, the present disclosure provides a kit comprising the nanoparticles of the present disclosure.

[0417] The kit may further include packaging materials and instructions for use to form a pharmaceutical composition and / or a delivery agent, which may include saline, a buffer, or a lipidoid.

[0418] 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 an empty lipid nanoparticle composition and a second container that includes a solution having 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.

[0419] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise specified herein, each of the following terms shall have the meaning indicated below. Further definitions are set forth throughout this application.

[0420] The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one member of the group, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0421] 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 term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "plural."

[0422] Furthermore, as used herein, "and / or" contemplates the specific disclosure of each of two particular features or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0423] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0424] Units, prefixes, and symbols are indicated in the form recognized by the 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 between the upper and lower limits of the range, and each fraction thereof, 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 where either, neither, or both limits are included is also encompassed by the disclosure. When values ​​are explicitly listed, it is understood that values ​​of approximately the same quantity or amount as the listed value are also included within the scope of the disclosure. When a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is included within the scope of the disclosure. Conversely, when different elements or groups of elements are individually disclosed, the combination is also disclosed. Where any element of a disclosure is disclosed with multiple options, examples of that disclosure in which each option is excluded alone or in any combination with the other options are also disclosed herein. Multiple elements of a disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0425] The term "about" as 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 ordinary skill in the art, for example, an interval of accuracy of ±10%.

[0426] When ranges are specified, the endpoints are included. Additionally, unless otherwise stated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed in ranges can be taken to be up to one-tenth of the unit of the lower limit of the range for any particular value or subrange within the ranges defined in different embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0427] 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 a certain interval such that the effects of each agent on the patient may overlap. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the administration of the agents is sufficiently close together that a combined (e.g., synergistic) effect is achieved.

[0428] As used herein, the term "CFTR" refers to the cystic fibrosis transmembrane conductance regulator, which is the main gene associated with cystic fibrosis. See NM_000492, NP_000483, XM_011515751, XP_011514053, XM_011515752, XP_011514054, XM_011515753, XP_011514055, XM_011515754, XP_011514056. CFTR is also known as ATP-binding cassette subfamily C, member 7 ("ABCC7"). CFTR is an enzyme (EC3.6.3.49) that plays a key role in transport pathways and functions as a chloride ion channel. Lack of functional CFTR prevents chloride ion excretion and increases sodium ion absorption. Welsh, MJ et al., J. Clin. Invest. 80:1523-1526 (1987). This causes water to move from the mucus into the cell, producing a viscous mucus. CFTR is localized in the cytoplasm, endosomes, extracellular space, and cell membrane of cells. The protein is 1480 amino acids long. Complete or partial loss of CFTR function results in thick and sticky mucus, which causes difficulty in breathing, indigestion, and shortens life span.

[0429] 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), enantiomer, or diastereomer 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 atomic nuclei. For example, isotopes of hydrogen include tritium and deuterium. Additionally, the compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules by conventional methods to form solvates and hydrates.

[0430] As used herein, the term "deliver" or "delivering" refers to providing an entity to a destination. For example, delivering a nucleic acid, such as an mRNA, to a subject can include administering a nanoparticle composition comprising the nucleic acid to the subject, for example, by any of the means described herein for administering a lipid nanoparticle composition described herein to a subject.

[0431] 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.

[0432] 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, for example, the amount of mRNA expressing the protein in an amount sufficient to improve, reduce, eliminate, or prevent the signs and symptoms associated with the protein deficiency, as compared to the severity of the symptoms observed without administering the agent. The term "effective amount" may be used interchangeably with "effective dose", "therapeutically effective amount", or "therapeutically effective dose".

[0433] As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of the nanoparticle composition relative to the initial total amount of polynucleotide used in the nanoparticle composition. For example, if 97 mg of polynucleotide is encapsulated in the nanoparticle composition out of a total of 100 mg of polynucleotide initially provided in the composition, the encapsulation efficiency can be given as 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial surrounding, enclosing, surrounding, or covering.

[0434] As used herein, "airway epithelium" refers to the layer of cells lining the conducting airways of a patient and plays an important role in protecting the alveoli, where gas exchange occurs, from damage. The airway epithelium serves to remove and neutralize potentially harmful substances from inhaled air.

[0435] 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); and (3) translation of the mRNA into a polypeptide or protein, as well as post-translational modification of the polypeptide or protein.

[0436] As used herein, the terms "linker," "linker structure," and "linker moiety" refer to a group of atoms, e.g., 10 to 1,000 atoms, which may consist of atoms or groups, such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine.

[0437] In some embodiments, the linker can be attached to a modified nucleoside or nucleotide of the nucleic acid base or sugar moiety at a first end, and to a payload, such as a detectable or therapeutic drug, at a second end. The linker can be of sufficient length so as not to prevent incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form a polynucleotide multimer (e.g., via the linkage of two or more chimeric polynucleotide molecules or IVT polynucleotides) or a polynucleotide conjugate, 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 may 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, such as 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 within the linker, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved using, for example, tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or photolysis, and ester bonds, which can be cleaved by, for example, acid or base hydrolysis.

[0438] In other embodiments, a "linker" can be a portion of a cationic lipid, such as a sterol amine. In this context, the linker can serve to link the lipid moiety to the amine portion of the cationic lipid and can refer to a group of atoms, e.g., 5-100 atoms, which can consist of atoms or groups, such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine.

[0439] The term "lipid", as generally defined herein, refers to a small molecule with hydrophobic or amphiphilic properties. Lipids can be naturally occurring or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, and polyketides, as well as prenol lipids. In some cases, the amphiphilic properties of some lipids lead to their formation of liposomes, vesicles, or membranes in aqueous media.

[0440] As used herein, the term "lipid amine" refers to a lipid molecule to which one or more amine functional groups are added.The amine functional group 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.The lipid amine can include sterol amines, where the lipid portion of the molecule is a steroid, such as cholesterol or a related moiety.

[0441] As used herein, the phrase "a moiety cleavable under physiological conditions" refers to, for example, an ester, amide, carbonate, carbamate, or urea moiety.

[0442] As used herein, "patient" refers to a subject (e.g., a human subject) who is seeking or in need of treatment, who is in need of treatment, who is receiving treatment, who will be receiving treatment, or who is receiving medical care for a particular disease or condition by a trained professional.

[0443] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0444] As used herein, the phrase "pharmaceutical acceptable additive" refers to any ingredient other than the compounds described herein (e.g., a medium capable of suspending or dissolving the active compound) that has substantially non-toxic and non-inflammatory properties in patients.Additives can include, for example, anti-adhesive agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (coloring agents), emollients, emulsifiers, excipients (diluents), film-forming agents or coatings, flavors, flavorings, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water.

[0445] The present disclosure also includes salts of the compounds described herein. As used herein, "salt" refers to derivatives of the compounds of the present disclosure, 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 an appropriate 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, 17th Edition, 1999, pp. 171-175, 1999. thed., 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.

[0446] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, 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"). It also includes modified forms, for example, by alkylation and / or capping of polynucleotides, as well as unmodified forms. More specifically, the term "polynucleotide" includes polyribonucleotides (containing 2-deoxy-D-ribose), whether spliced ​​or not, including polydeoxyribonucleotides (containing D-ribose), tRNA, rRNA, hRNA, siRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers that contain normucleotidic backbones, such as polyamides (e.g., peptide nucleic acid ("PNA")) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers that contain nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA. In certain embodiments, the polynucleotide comprises an mRNA. In other embodiments, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are replaced with non-natural nucleobases (e.g., all uridines of the polynucleotides disclosed herein can be replaced with non-natural nucleobases, such as 5-methoxyuridine). In some embodiments, the polynucleotides (e.g., synthetic RNA or DNA) contain only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) for synthetic DNA, and A, C, G, and U (uridine) for synthetic RNA.

[0447] Those skilled in the art will understand that the T bases of the codon maps disclosed herein are present in DNA, and in the corresponding RNA, the T bases are replaced by U bases. For example, the codon nucleotide sequences disclosed herein in DNA form, such as vectors or in vitro translation (IVT) templates, have their T bases transcribed as U bases in the corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (including T) and 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 generated 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 with U replaced with pseudouridine).

[0448] Canonical AT and GC base pairs occur under conditions that allow the formation of hydrogen bonds 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 nucleoside bases that do not efficiently form canonical base pairs 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 does base pair with isoguanosine (U.S. Pat. No. 5,681,702, Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the methods described in Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the methods 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 in Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the methods described in Collins et al., U.S. Pat. No. 5,780,610. 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.

[0449] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymers 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 in the definition are polypeptides that contain, for example, one or more analogs of amino acids (e.g., unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine, etc.), as well as other modifications known in the art.

[0450] 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 above. Polypeptides can be monomers or multi-molecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can 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" can be 50 amino acids or less in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0451] As used herein, the term "preventing" 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, characteristics, 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, characteristics, or manifestations 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 onset of a pathology associated with the infection, disease, disorder, and / or condition.

[0452] As used herein, "prophylactic" refers to a treatment or course of action used to prevent the onset, progression, or spread of a disease.

[0453] "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, livestock animals, farm animals, zoo animals, sport animals, pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, primates, such as apes, 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.

[0454] As used herein, the term "substantially" refers to a qualitative state of exhibiting the full or nearly full extent or degree of a desired feature or characteristic. Those skilled in the art of biology will understand that biological and chemical properties rarely progress to perfection and / or completeness or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical characteristics.

[0455] 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.

[0456] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, therapeutic, diagnostic, prophylactic, etc.) delivered that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, ameliorate the signs and symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0457] As used herein, the term "treating" or "treatment" or "therapy" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the 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 alleviating signs and symptoms associated with the disease, increasing the lifespan of a patient (improving survival rate), reducing the severity of the disease, preventing or delaying the onset of the disease, etc. Treatment may be administered to subjects who do not show signs of a disease, disorder, and / or condition and / or to subjects who only show early symptoms of a disease, disorder, and / or condition, with the aim of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition.

[0458] 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).

[0459] As used herein, the term "alkylene" refers to a linking alkyl group.

[0460] 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.

[0461] 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.

[0462] As used herein, the terms "carbocycle", "carbocyclyl" and "carbocyclic group" are synonymous and refer to a monocyclic or polycyclic 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.

[0463] As used herein, the term "cycloalkyl" refers to non-aromatic carbocycles and represents a subset of carbocycles. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0464] As used herein, the term "carbocyclylene" refers to a linking carbocyclyl group.

[0465] "C 3-6 The term "carbocycle" means a carbocycle containing a single ring having from 3 to 6 carbon atoms. A carbocycle can contain one or more double bonds and can be aromatic (e.g., an aryl group). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. A carbocycle can be optionally substituted.

[0466] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group. An example of a carbocyclylalkyl group is benzyl.

[0467] As used herein, the term "heterocycle", "heterocyclyl" or "heterocyclic group" refers to a monocyclic or polycyclic 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.

[0468] As used herein, the term "heterocycloalkyl" refers to non-aromatic heterocycles and represents a subset of heterocycles. Examples of heterocycloalkyl groups include azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, and the like.

[0469] As used herein, the term "heterocyclylene" refers to a linking heterocyclyl group.

[0470] 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.

[0471] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group.

[0472] As used herein, the term "arylene" refers to a linking aryl group.

[0473] As used herein, a "heteroaryl group" is a heterocyclic group that contains one or more heterocyclic aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted.

[0474] As used herein, the term "heteroarylene" refers to a linking heteroaryl group.

[0475] 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). Examples of oxygen protecting groups can include optionally substituted alkyl, carbocyclyl, heterocyclyl, carbocyclylalkyl, and heterocyclylalkyl groups.

[0476] 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).

[0477] Alkyl, alkenyl, alkynyl, and cyclyl (eg, carbocyclyl and heterocyclyl) groups may be optionally substituted, unless otherwise specified. The optional substituents can be 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., C(O)R, alternatively represented as 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. R is an alkyl, alkenyl, or alkynyl group as defined herein.

[0478] 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 limited to the above description, but is as set forth in the appended claims.

[0479] When ranges are specified, the endpoints are included. Additionally, unless otherwise stated or apparent from the context and the understanding of one of ordinary skill in the art, it is understood that values ​​expressed in ranges can be taken to be any particular value or subrange within the ranges defined in different embodiments of this disclosure, down to one tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0480] Furthermore, it is understood that any particular embodiment of the present disclosure that falls within the scope of the prior art may be expressly excluded from any one or more claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein, since they are 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 claims for any reason, whether related to the existence of prior art or not.

[0481] It is 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.

[0482] All sources cited herein, e.g., references, publications, databases, database entries, and techniques, are incorporated by reference into this application, even if not expressly stated in the citation. In the event of a conflict between a cited source and the statement in this application, the statement in this application shall control.

[0483] Section and table headings are not intended to be limiting. EXAMPLES

[0484] Example 1 Generation of empty lipid nanoparticles Empty lipid nanoparticles were prepared according to the process outlined in Figure 1. Lipids (ionic lipid:DSPC:cholesterol:DMG-PEG 2000 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 in mixer 1 and mixer 2, and a lipid:buffer (25% ethanol) volume ratio of 1:3 in mixer 3. After a 5 second residence time, 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) into 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 subsequently added.

[0485] Example 2 Particle size comparison Lipids (ionic lipid:DSPC:cholesterol:DMG-PEG 2000 lipid) were dissolved in ethanol at a concentration of 24 mg / mL (40 mM 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).

[0486] The lipid solution was delivered at 2.5 mL / min and the acidified aqueous buffer stream was delivered at 7.5 mL / min. The two streams were mixed using a 0.5 mm ID mixing tee. The lipid concentration after nanoprecipitation was 6 mg / mL. 600 mL of LNP solution was made for each sample.

[0487] A 30 kDa mPES filter was used for sample number 1 and a 100 kDa mPES filter was used for sample number 2. A 5-fold volume ultrafiltration (UF1) was performed first, followed by a 5-fold volume diafiltration (DF) for the 30 kDa filter or an 8-fold volume diafiltration (DF) for the 100 kDa filter. The final step is a further 8-fold ultrafiltration (UF2).

[0488] See Table 2-A for final lipid concentrations and calculated yields for each sample.

[0489] 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 make a final product of 74.5 mg / mL LNPs and 200 mg / mL sucrose. [Table 2]

[0490] The average diameter of the empty lipid nanoparticles prepared at pH 4 and pH 5 as described above 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 3]

[0491] Example 3 Characterization of empty lipid nanoparticles The average diameter of empty lipid nanoparticles prepared according to the process of Example 2, except as described below, was measured.

[0492] The average diameter 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.

[0493] The average diameter 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, and show that higher buffer concentrations favor the formation of smaller sized particles.

[0494] The average diameter of empty lipid nanoparticles was compared over 25 hours at different 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 show that high buffer concentration favors the formation of small sized particles that remain small over 25 hours.

[0495] The average diameter of empty lipid nanoparticles was compared at different pH over a period of 25 hours. The average particle size was measured by DLS. The results are shown in Figure 5 and show that low pH favors the formation of small sized particles that remain small over a period of 25 hours.

[0496] The zeta potential of the empty lipid nanoparticles prepared according to the process of Example 2 was measured with a Mobius zeta potential instrument from Wyatt Technologies. This instrument characterizes the 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, showing high zeta potential at low pH, which is almost independent of buffer concentration and lipid solution concentration.

[0497] 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.

[0498] Example 4 Preparation of loaded lipid nanoparticles Empty lipid nanoparticles prepared according to Example 2, Figure 1, were loaded with nucleic acid (mRNA) according to the process shown in Figure 8. The loading of mRNA was performed using a post-loading (PHL) process. eLNPs at a lipid concentration of 11.72 mg / mL in 5 mM acetic acid (pH 5) and 75 g / L sucrose were mixed with 42.5 mM sodium acetate pH 5.0 containing mRNA at a concentration of 1.0 mg / mL. The eLNP solution and mRNA were mixed using a multi-inlet vortex mixer in a volume ratio of 3:2 eLNP:mRNA. After loading the eLNPs with mRNA, they were mixed in-line with a neutralization buffer containing 120 mM TRIS pH 8.12 in a nanoparticle:buffer volume ratio of 5:1 after a residence time of 60 seconds. After this addition step, the nanoparticle formulation was mixed in-line with a buffer containing 20 mM TRIS (pH 7.5), 1.42 mg / mL DMG-PEG 2000, 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 with 300 nM NaCl solution in running buffer (20 mM TRIS, 14.3 mM sodium acetate, and 32 g / L sucrose, pH 7.5) 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 strength of approximately 1 mg / mL (e.g., 0.5-2 mg / mL).

[0499] Example 5 Characterization of loaded lipid nanoparticles Loaded lipid nanoparticle compositions were prepared at different mRNA stock concentrations according to the process described in Example 4. For process details, see Table 5-A below. [Table 4]

[0500] The obtained mean particle size (measured by DLS) and polydispersity (PDI) values ​​are compared in Figure 9. These values ​​were calculated using cumulant analysis. The diameter in the MP column was measured after the PI buffer step. The mean particle size was consistently 75-85 nm. The encapsulation efficiency was greater than 98%. The loading mRNA concentration had little effect on the particle size.

[0501] Example 6 Alternative generation of empty lipid nanoparticles Empty lipid nanoparticles were prepared according to the process outlined in Figure 11. Lipids (ionic lipid:DSPC:cholesterol:DMG-PEG 2000 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 a volumetric ratio of 5:7 eLNP:buffer. The resulting diluted eLNPs were then buffer exchanged and concentrated using tangential flow filtration (TFF) into a final buffer containing 37.5 mM sodium acetate at pH 4. A solution of 70% sucrose in 37.5 mM acetate buffer pH 4 was then subsequently added.

[0502] 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. The loading of mRNA was performed using a post-loading (PHL) process. 32.5 mM acetic acid pH 5 containing mRNA was added using dialysis. The concentration was measured using NaOH digestion. The buffer was used to confirm the concentration of acetic acid and sodium acetate in 37.5 mM acetate buffer at pH 4, 4.5, 5, 5.5, or 6. This specially concentrated buffer solution was used to dilute the water containing mRNA (additional water) to 1.6 mg / mL of mRNA at the respective pH of the 37.5 mM buffer. A 37.5 mM acetic acid and 20% sucrose solution at pH 4, 4.5, 5, 5.5, or 6 of eLNP with lipid concentration of 37.25 mg / mL was mixed with 37.5 mM sodium acetate with mRNA concentration of 1.6 mg / mL at the same pH as the eLNP solution. The eLNP solution and mRNA were mixed using a multi-inlet vortex mixer at a volumetric ratio of 1:2.5 eLNP:mRNA. After loading the eLNPs with mRNA, they were mixed in-line with a neutralization buffer containing TRIS buffer and 32.3% sucrose after a residence time of 60 seconds. 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-PEG 2000.

[0503] 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 loaded lipid nanoparticles measured before mixing at different loading pH of blank lipid nanoparticles and mRNA solution. 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 5]

[0504] Table 8-B shows a comparison of the encapsulation efficiency of loaded lipid nanoparticles prepared using similar procedures outlined in Examples 6 and 7, except that the acidified buffer had a pH of 5. Figure 14 shows the average diameter in nm of loaded lipid nanoparticles at different loading pHs of blank lipid nanoparticles and mRNA solutions measured before mixing. [Table 6]

[0505] N to P is the ratio of nitrogen to phosphorus in the nanoparticles.

[0506] Example 9 Alternative 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 15. The loading of mRNA was performed using a post-loading (PHL) process. eLNPs at a lipid concentration of 2.93 mg / mL in 5 mM acetic acid (pH 5) and 7.5% sucrose were mixed with mRNA at a concentration of 0.25 mg / mL in 42.5 mM sodium acetate pH 5.0. The eLNP solution and mRNA were mixed using a multi-inlet vortex mixer in a volume ratio of eLNP:mRNA of 3:2. After loading the eLNPs with mRNA, they were mixed in-line with a neutralization buffer containing 120 mM TRIS pH 8.12 in a nanoparticle:buffer volume ratio of 5:1 after a residence time of 60 seconds. After this addition step, the nanoparticle formulation was mixed in-line with a buffer containing 20 mM TRIS (pH 7.5), 0.363 mg / mL DMG-PEG 2000, and 0.625 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 with 300 nM NaCl solution in running buffer (20 mM TRIS, 14.3 mM sodium acetate, and 32 g / L sucrose, pH 7.5) 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 strength of approximately 1 mg / mL (e.g., 0.5-2 mg / mL).

[0507] Example 10 Alternative 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 16. The loading of mRNA was performed using a post-loading (PHL) process. eLNPs at a lipid concentration of 24.6 mg / mL in 5 mM acetic acid (pH 5) and 20% sucrose were mixed with mRNA at a concentration of 0.56 mg / mL in 26 mM sodium acetate pH 5.0. The eLNP solution and mRNA were mixed using a multi-inlet vortex mixer in a volume ratio of 3:2 eLNP:mRNA. After loading the eLNPs with mRNA, they were mixed in-line after a residence time of 60 seconds with a neutralization buffer containing 120 mM TRIS pH 8.3 and 16.2% sucrose in a nanoparticle:buffer volume ratio of 5:1. After this addition step, the nanoparticle formulation was mixed in-line with a buffer containing 20 mM TRIS, pH 7.5, 1.452 mg / mL DMG-PEG 2000. The resulting nanoparticle suspension was concentrated using tangential flow filtration (TFF) and diluted with 300 nM NaCl solution in running buffer (20 mM TRIS, 14.3 mM sodium acetate, and 2.5 mg / mL sterol amine, pH 7.5) to a final buffer matrix containing 140 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 strength of approximately 1 mg / mL (e.g., 0.5-2 mg / mL).

[0508] 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 a lipid nanoparticle composition, comprising: (a) (i) an ionic lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid are mixed with an aqueous buffer solution having a pH of about 4.5 or less to obtain an empty lipid nanoparticle composition, and (b) mixing the empty lipid nanoparticle composition with a payload to form a lipid nanoparticle composition filled with the payload, wherein the payload is for delivery to epithelial cells, and (c) adding a cationic agent to the lipid nanoparticle composition filled with the payload, wherein the process includes that the cationic agent is a cationic lipid.

2. The process according to claim 1, wherein the payload contains a nucleic acid, and the nucleic acid is mRNA.

3. The process according to claim 2, wherein the nucleic acid is provided as a nucleic acid solution containing (i) the nucleic acid and (ii) a buffer capable of maintaining an acidic pH.

4. The process according to any one of claims 1 to 3, wherein the mixing in step (b) is carried out at a pH of about 5 to about 6.

5. The process according to claim 1, wherein the cationic lipid is a sterol amine containing a sterol-based hydrophobic moiety and a hydrophilic moiety.

6. The sterol amine is a compound of formula A2a: 【Chemical Formula 1】 or a salt thereof, wherein ---- is a single bond or a double bond, R １ is C １－１４ alkyl or C １－１４ alkenyl, and L ａ is absent, -O-, -S-S-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH ２ -NH-C(O)-, -C(O)O-, -OC(O)-CH ２ -CH ２ -C(=O)N-, -S-S-CH ２ , -SS-CH ２ -CH ２ -C(O)N-, or formula (a): 【Chemical 2】 is a group of, Y １ is C １－１０ alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), or C １－６ alkyl-(5- to 6-membered heteroaryl), and Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) contain 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) are each optionally C １－６ alkyl, halo, OH, O(C １－６ alkyl), C １－６ alkyl-OH, NH ２ , NH(C １－６ alkyl), N(C １－６ alkyl) ２ , 3- to 8-membered heterocycloalkyl (optionally substituted with 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, C １－１４ alkyl), 5- to 6-membered heteroaryl, NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl), and are substituted with 1, 2, 3, or 4 substituents selected from n = 1 or 2. The process according to claim 5.

7. The sterol amine is a compound of formula A3a: 【Chemical Formula 3】 or a salt thereof, wherein ---- is a single bond or a double bond, R ２ is H or C １－６ alkyl, and L ａ is absent, -O-, -S-S-, -OC(=O)-, -C(=O)N-, -OC(=O)N-, CH ２ -NH-C(O)-, -C(O)O-, -OC(O)-CH ２ -CH ２ -C(=O)N-, -S-S-CH ２ , -SS-CH ２ -CH ２ -C(O)N-, or formula (a): 【Chemical Formula 4】 is a group of, Y １ is C １－１０ alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), or C １－６ alkyl-(5- to 6-membered heteroaryl), and Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) are each optionally C １－６ alkyl, halo, OH, O(C １－６ alkyl), C １－６ alkyl-OH, NH ２ , NH(C １－６ alkyl), N(C １－６ alkyl) ２ , 3- to 8-membered heterocycloalkyl (optionally substituted with 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, C １－１４ alkyl), 5- to 6-membered heteroaryl, NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl), and are substituted with 1, 2, 3, or 4 substituents selected from n = 1 or 2. The process according to claim 5.

8. The sterol amine is a compound of formula A4: 【Chemical Formula 5】 or a salt thereof, wherein Z １ is OH or C ３－６ alkyl, and L is absent, or is —O—, —S—S—, —OC(═O), —C(═O)N—, —OC(═O)N—, CH ２ —NH—C(O)—, —C(O)O—, —OC(O)—CH ２ —CH ２ —C(═O)N—, —S—S—CH ２ or —SS—CH ２ —CH ２ —C(═O)N—, and Y １ is C １－１０ alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), or C １－６ alkyl-(5- to 6-membered heteroaryl), and Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, Here, the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C １－６ alkyl-(3- to 8-membered heterocycloalkyl), and C １－６ alkyl-(5- to 6-membered heteroaryl) are each optionally C １－６ alkyl, halo, OH, O(C １－６ alkyl), C １－６ alkyl-OH, NH ２ , NH(C １－６ alkyl), N(C １－６ alkyl) ２ , 3- to 8-membered heterocycloalkyl (optionally substituted with 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, and C １－１４ alkyl), 5- to 6-membered heteroaryl, NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl), and are substituted with 1, 2, 3, or 4 substituents selected from n = 1 or 2. The process according to claim 5.

9. Y １ is 【Chemical Formula 6-1】 (28) N(CH ３ ) ２ ; 【Chemical Formula 6-2】 The process according to any one of claims 6 to 8, selected from

10. The sterol amine is a compound of formula A5: 【Chemical Formula 7】 or a salt thereof, wherein Z ２ is OH or isopropyl, L ３ is —CH ２ —NH—C(O)—, —C(O)NH—, or —C(O)O—, the process according to claim 5.

11. The sterol amine is 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 a compound selected from, or a salt thereof. The process according to claim 5.

12. The sterol amine is SA3: 【Chemical Formula 8】 or a salt thereof. The process according to claim 5.

13. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein the cationic agent is provided as a cationic agent solution containing the cationic agent and a buffer.

14. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, further comprising adding a surfactant to the filled lipid nanoparticles.

15. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, further comprising one or more further steps selected from the following: diluting the composition with a dilution buffer; adjusting the pH of the composition; filtering the composition; concentrating the composition; exchanging the buffer of the composition; and adding an osmotic pressure regulator to the composition.

16. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein the aqueous buffer solution in step (a) has a pH of about 3.5 to about 4.

5.

17. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein step (a) further comprises one or more further 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 of the composition; and adding a cryoprotective substance to the composition.

18. The ionic lipid is a compound of formula (I): 【Chemical Formula 9】 or an N-oxide or salt thereof, wherein R １ is 【Chemical Formula 10】 and 【Chemical 11】 represents a point of attachment, 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 each independently is selected from C １－１４ alkyl and C ２－１４ alkenyl, R ４ is -(CH ２ ) ｎ OH and 【Chemical Formula 12】 is selected from n is selected from 1, 2, 3, 4, and 5, 【Chemical 13】 represents a point of attachment, 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 n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, 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 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, and I is selected from 1, 2, 3, 4, and 5, m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, in the process according to any one of claims 1 to 3, 5 to 8 and 10 to 12.

19. The ionic lipid has a structure: 【Chemical Formula 14】 or an N-oxide or salt thereof, in the process according to any one of claims 1 to 3, 5 to 8 and 10 to 12.

20. 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 (DUPPC), 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 The process according to any one of claims 1 to 3, 5 to 8, and 10 to 12, selected from sphingomyelin.

21. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein 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.

22. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein 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.

23. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein the lipid solution, empty lipid nanoparticle composition, or loaded lipid nanoparticle composition contains about 30 mol% to about 60 mol% of an ionic lipid, about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 50 mol% of a structural lipid, and about 0.1 mol% to about 2 mol% of a PEG-lipid, based on the total lipid.

24. The process according to any one of claims 1 to 3, 5 to 8 and 10 to 12, wherein the weight ratio of the cationic agent to the payload is about 1:1 to about 4:

1.

25. A lipid nanoparticle composition prepared by the process according to any one of claims 1 to 3, 5 to 8 and 10 to 12.

26. A pharmaceutical composition comprising the lipid nanoparticle composition according to claim 25 and at least one pharmaceutically acceptable additive.

Images