Method of producing lipid-encapsulated RNA nanoparticles

Abstract

To provide a method for forming lipid-encapsulated RNA nanoparticles that optimizes particle size and uniformity, and enables large-scale production.SOLUTION: A method of producing lipid-encapsulated RNA nanoparticles, comprises the steps of: a) flowing an aqueous solution comprising RNA through a first tube having an inner diameter (ID) of 0.1 inch to 0.132 inch; b) flowing an ethanol solution comprising lipids through a second tube having an ID of 0.005 inch to 0.02 inch at one third the flow rate of the aqueous solution through the first tube, wherein the lipids comprise a cationic lipid; and c) mixing the ethanol solution with the aqueous solution by flowing the ethanol solution and the aqueous solution into a mixing module consisting of the second tube perpendicularly joined to the first tube, wherein the mixing step produces an output solution flowing in the first tube comprising a turbulent flow of the RNA and the lipids in about 10 v / v% to 75 v / v% ethanol, and wherein the lipid-encapsulated RNA nanoparticles have a bilayer structure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 820,496, filed March 19, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Lipids are used as materials for ribonucleic acid (RNA) delivery due to their ability to form lipid nanoparticles that encapsulate RNA for delivery to target cells upon parenteral administration (Zimmermann, 2006, Nature, doi:10.1038 / nature04688).

[0003] Various methods for producing lipid-encapsulated RNA nanoparticles are known. For example, WO 2001 / 005373 discloses a technique for producing lipid-encapsulated RNA nanoparticles using an ethanol injection-type manufacturing method using a static mixer that provides a turbulent flow environment, whereby vesicles are combined with therapeutic molecules after formation. US 2004 / 0142025 discloses a technique for forming lipid-encapsulated RNA nanoparticles using non-turbulent mixing and a series of continuous stepwise dilutions. US 6,843,942 discloses a non-turbulent mixing method in which particles are formed by spraying lipids in an organic solution pipe through an orifice onto nucleic acid in an aqueous solution flowing through an orifice. discloses the encapsulation of siRNA in lipid nanoparticles (LNPs) using turbulent mixing, in which lipids and RNA as counterflows enter a T-shaped mixing chamber from opposite arms at approximately the same speed, producing a 45-60% ethanol solution containing vesicles, which is then collected and further diluted (direct dilution method). US 9,404,127 discloses that the majority of LNPs purified by direct dilution have a non-lamellar morphology, ie, a non-bilayer structure.

[0004] There is a need for improved manufacturing methods and equipment for forming lipid-encapsulated RNA nanoparticles, including the ability to reliably scale up to large-scale production while optimizing particle size and uniformity. Summary of the Invention

[0005] A method for producing lipid-encapsulated RNA nanoparticles is disclosed, comprising the steps of: a) flowing an aqueous solution containing RNA through a first tube having an inner diameter (ID) of approximately 0.1 to 0.132 inches; b) flowing an ethanol solution containing lipids through a second tube having an ID of approximately 0.005 to 0.02 inches at a flow rate one-third that of the aqueous solution passing through the first tube, wherein the lipids comprise cationic lipids; and c) mixing the ethanol solution with the aqueous solution by flowing them through a mixing module consisting of a second tube joined perpendicularly to the first tube, wherein the mixing step results in an exit solution flowing through the first tube comprising a turbulent flow of RNA and lipids in approximately 10% to 75% v / v ethanol, and the lipid-encapsulated RNA nanoparticles have a bilayer structure.

[0006] In some embodiments, the output flow rate is at least 200 ml / min. In another embodiment, the combined stream has a Reynolds number of at least 2,000.

[0007] In some embodiments, the aqueous solution is pumped through the first tubing by a first HPLC pump, preferably having a back pressure of at least 10 psi, 25 psi, 50 psi, 75 psi, or 100 psi. Preferably, the first tubing has an ID of 0.132 inches, and the aqueous solution is pumped at a flow rate of at least 30 ml / min, 45 ml / min, 60 ml / min, 75 ml / min, 90 ml / min, 105 ml / min, 120 ml / min, 150 ml / min, 225 ml / min, 262.5 ml / min, 300 ml / min, or 450 ml / min.

[0008] In some embodiments, the ethanol solution is pumped through the second tubing by a second HPLC pump, preferably with a back pressure of at least 40 psi, 80 psi, 150 psi, 300 psi, or 400 psi. Preferably, the second tubing has an ID of 0.007 inches, 0.01 inches, or 0.02 inches; and the ethanol solution is pumped at a flow rate of at least 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 50 ml / min, 60 ml / min, or 75 ml / min, 87.5 ml / min, 100 ml / min, or 150 ml / min.

[0009] Preferably, the aqueous and ethanolic solutions are maintained at 15 to 20°C.

[0010] In some embodiments, the mixing module comprises a second tube attached perpendicular to the first tube, the first tube having an opening through its wall, the opening being the same size as the outer diameter of the second tube, and the second tube mating with the opening so that the second solution in the second tube can be transferred continuously to the first solution in the first tube. The mixing module is preferably comprised of stainless steel tubing.

[0011] In some embodiments, the disclosed method further comprises pumping the dilution buffer through the third tube and introducing the dilution buffer into the discharge solution at the Y-connector region to mix the dilution buffer with the discharge solution to produce a diluted discharge solution. Preferably, the dilution buffer comprises 15 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5; 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5; 50 mM phosphate, pH 6.0; 20 mM HEPES, 50 mM NaCl, 9% sucrose, pH 7.4; or 50 mM HEPES, 50 mM NaCl, 9% sucrose, pH 7.4. Preferably, the diluted discharge solution comprises 6.25% ethanol; 8.25% ethanol; or 12.5% ethanol.

[0012] In some embodiments, the Y connector is at an angle of about 45°. Preferably, the Y connector is made of polyetheretherketone (PEEK).

[0013] In some embodiments, the dilution buffer is pumped through the third tubing at a flow rate of 400-900 mL / min, and the third tubing has an ID of 0.25 inches.

[0014] In another aspect, lipid-encapsulated RNA nanoparticles produced by the methods of the present disclosure are disclosed.

[0015] In another embodiment, lipid-encapsulated RNA nanoparticles containing RNA are disclosed, the lipid-encapsulated RNA nanoparticles being produced by a manufacturing method comprising the steps of: a) flowing an aqueous solution containing RNA through a first tube having an inner diameter (ID) of approximately 0.1 to 0.132 inches; b) flowing an ethanol solution containing lipids through a second tube having an ID of approximately 0.005 to 0.02 inches at a flow rate one-third that of the aqueous solution through the first tube, the lipids comprising cationic lipids; and c) mixing the ethanol solution with the aqueous solution by flowing them through a mixing module consisting of a second tube joined perpendicularly to the first tube, wherein the mixing step produces an output solution flowing through the first tube comprising a turbulent flow of RNA and lipids in approximately 10% to 75% v / v ethanol. The lipid-encapsulated RNA nanoparticles produced by the manufacturing method described herein have a bilayer structure, i.e., a lamellar morphology. Preferably, the lipid-encapsulated RNA nanoparticles have an average particle size of less than 70 nm, 80 nm, 90 nm, or 100 nm; a polydispersity index (PDI) of less than 0.09, 0.07, or 0.05; and encapsulation of greater than 94%, 96%, or 98% of RNA. Preferably, batch sizes are from 0.05 to at least 30 g RNA, and the variation in average particle size between batches is less than 10%.

[0016] In yet another embodiment, an apparatus for producing lipid nanoparticles comprising RNA, the apparatus comprising: a first tube having an ID of about 0.1 inches to 0.132 inches and connected at one end to a first HPLC pump and at the other end to a mixing module, the first HPLC pump configured to pump an aqueous solution comprising RNA through the first tube at a flow rate of at least 150 ml / min; a reservoir connected to the first HPLC pump, the first reservoir containing the aqueous solution; a first reservoir having an ID of about 0.005 inches to 0.02 inches and connected at one end to a second HPLC pump and at the other end to a mixing module. a second tube connected to the mixing module, wherein a second HPLC pump is configured to pump an ethanol solution containing lipids through the second tube at a flow rate greater than 50 ml / min, the second tube being perpendicularly connected to the first tube at the mixing module; a second reservoir connected to the second HPLC pump, the second reservoir containing the ethanol solution, the device being configured to mix the ethanol solution with the aqueous solution by introducing the ethanol solution into the aqueous solution at a region within the mixing module to generate an output solution; and the flow of the output solution generates turbulence.

[0017] In some embodiments, the devices described herein preferably have a mixing module in which the second tube extends partially through the wall of the first tube into the interior of the first tube; or the second tube extends up to the wall of the first tube and joins to the first tube. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 shows a flow chart of one embodiment of a method for producing lipid nanoparticles. Lipids were dissolved in ethanol, and RNA was dissolved in an acidic buffer solution (e.g., citrate buffer), both of which were filter-sterilized. The solutions were mixed to form particles according to the methods described herein, and PDI and particle size (PS) were analyzed. The particles were concentrated and purified by tangential flow filtration (TFF) to remove ethanol and unbound RNA, and PDI and PS were again monitored. The particle concentration was then adjusted according to the measured total RNA concentration. The particles were filter-sterilized, filled, polished, and frozen.

[0019] [Figure 2] FIG. 2 shows a table of exemplary lipid RNA formulations, including particle sizes.

[0020] [Figure 3] FIG. 3 shows a table of exemplary lipid RNA formulations, including particle sizes and batch sizes.

[0021] [Figure 4]Figure 4 shows an apparatus for producing lipid-encapsulated RNA nanoparticles. An aqueous solution containing RNA is transported by an HPLC pump through tubing comprising 0.03-inch ID PEEK tubing, 0.05-inch ID PEEK connectors, 0.0625-inch silicone tubing, 0.122-inch ID silicone tubing, and a 0.132-inch ID stainless steel section. An organic solution containing lipids is transported by an HPLC pump through tubing comprising, for example, 0.03-inch ID PEEK tubing, 0.02-inch ID PEEK connectors, and a 0.01-inch ID stainless steel section. The organic solution is pumped into the aqueous solution at a 90° angle in the mixing zone. The 0.122-inch ID silicone tubing transports the mixed lipid-RNA output into 0.25-inch ID polypropylene tubing, which meets the dilution buffer at a 45° angle in the dilution zone. The tubing, which meets the dilution buffer at a 45° angle in the dilution zone, allows for serial dilutions to be performed using 1, 2, 3, or 4 dilution zones of 45° angle tubing. After the dilution step, the diluted particles are collected in a stainless steel jacketed vessel maintained at 15-20° C. The particles are further processed by tangential flow filtration using a diaphragm or centrifugal pump.

[0022] [Figure 5] Figure 5 shows the mixing module in more detail. Nucleic acids in buffer are transported through the inlet arm of a first (e.g., 0.100-0.132 inch ID) stainless steel tube. Lipids in ethanol are transported through a second (e.g., 0.005-0.010 inch ID) stainless steel tube attached perpendicular to the first tube. Holes in the wall of the first tube allow the transport of liquid from the second tube to the interior of the first tube. Lipid-encapsulated RNA nanoparticles resulting from mixing exit through the outlet arm of the first tube.

[0023] [Figure 6A] Figure 6A shows the scalability of the manufacturing method described in Example 1. Particle size remained below 80 nm for batches of 0.05 g, 0.5 g, 1 g, 3 g, 15 g, and 30 g RNA.

[0024] [Figure 6B] Figure 6B shows the reproducibility of lipid nanoparticles produced by the manufacturing method described in Example 1. Particle size remained below 80 nm in one 15 g batch and three 30 g batches.

[0025] [Figure 7] Figure 7 shows cryo-TEM images of lipid-encapsulated RNA nanoparticles. The image on the left shows RNA-loaded nanoparticles, most of which have a spherical, unilamellar structure. Magnification: 52,000x, Scale bar: 200 nm. The image on the right shows a higher magnification of 110,000x, Scale bar: 100 nm. DETAILED DESCRIPTION OF THE INVENTION

[0026] It is understood that various configurations of the subject technology will be readily apparent to those skilled in the art from the present disclosure, wherein various configurations of the subject technology have been shown and described by way of illustration. As will be recognized, the subject technology is capable of other different configurations, and its several details can be modified in various other respects, all without departing from the scope of the subject technology. Accordingly, the summary, drawings, and detailed description are to be regarded as illustrative in nature, and not as restrictive.

[0027] Applicants have discovered a method for producing lipid-encapsulated RNA nanoparticles using turbulent flow to mix lipids with single- or double-stranded ribonucleic acid (ssRNA or dsRNA) to provide nearly monodisperse particles less than 100 nm in size that have a lamellar morphology, i.e., contain a bilayer structure. The method is scalable to more than 30 g of RNA. Lipid-encapsulated RNA nanoparticles containing RNA produced by the method described herein are useful for in vitro RNA delivery and improve safety and efficacy upon intravenous administration in animal models.

[0028] In one embodiment, a method for producing lipid-encapsulated RNA nanoparticles comprises the steps of: a) flowing an aqueous solution containing RNA through a first tube having an inner diameter (ID) of about 0.1 inches to 0.132 inches; b) flowing an ethanol solution containing lipids through a second tube having an ID of about 0.005 inches to 0.02 inches at a flow rate one-third that of the aqueous solution through the first tube, wherein the lipids comprise cationic lipids; and c) mixing the ethanol solution with the aqueous solution by flowing the ethanol solution and the aqueous solution through a mixing module consisting of a second tube joined perpendicularly to the first tube, wherein the mixing step produces an output solution flowing through the first tube comprising a turbulent flow of RNA and lipids in about 10% v / v to 75% v / v ethanol, wherein the lipid-encapsulated RNA nanoparticles have a bilayer structure, and the lipids are represented by Formula I: [ka] (I) [In the formula, R 5 and R 6 are each independently a straight-chain or branched-chain C 1- C 31 Alkyl, C 2- C 31 Alkenyl or C 2- C 31 selected from the group consisting of alkynyl and cholesteryl; L 5 and L 6 are each independently a linear C 1- C 20 Alkyl and C 2- C 20 alkenyl; X 5 is -C(O)O- or -OC(O)-; X 6 is -C(O)O- or -OC(O)-; X 7 is S or O; L 7 is absent or lower alkyl; R 4 is a linear or branched chain C 1- C alkyl; and R 7 and R 8 are each independently hydrogen and a straight or branched chain C 1-C6 alkyl] or a pharmaceutically acceptable salt or solvate thereof.

[0029] In another aspect, lipid-encapsulated RNA nanoparticles produced by the methods provided herein are disclosed.

[0030] In yet another embodiment, we disclose lipid-encapsulated RNA nanoparticles produced by the following process: a) flowing an aqueous solution containing RNA through a first tube having an inner diameter (ID) of about 0.1 to 0.132 inches; b) flowing an ethanol solution containing lipids through a second tube having an ID of about 0.005 to 0.02 inches at a flow rate one-third that of the aqueous solution through the first tube, wherein the lipids comprise cationic lipids; and c) mixing the ethanol solution with the aqueous solution by flowing them through a mixing module consisting of a second tube joined perpendicularly to the first tube, wherein the mixing step produces an outlet solution flowing through the first tube comprising a turbulent flow of RNA and lipids in about 10% to 75% v / v ethanol, the lipid-encapsulated RNA nanoparticles having a bilayer structure, and the lipids comprise a cationic lipid represented by Formula I.

[0031] In some embodiments, the effluent solution flowing through the first tube comprises a turbulent flow of RNA and lipids in about 10% v / v to about 50% v / v ethanol.

[0032] In some embodiments, the effluent solution flowing through the first tube comprises a turbulent flow of RNA and lipids in about 16% v / v to about 50% v / v ethanol.

[0033] In some embodiments, the effluent solution flowing through the first tube comprises a turbulent flow of RNA and lipids in about 10 v / v%, 15 v / v%, 20 v / v%, 25 v / v%, 30 v / v%, 35 v / v%, 40 v / v%, 44 v / v%, 50 v / v%, 55 v / v%, 60 v / v%, 70 v / v% or 75 v / v% ethanol.

[0034] In some embodiments, the output has a flow rate of at least 200 ml / min.

[0035] In some embodiments, the exit flow rate has a Reynolds number of at least 2,000.

[0036] In some embodiments, the aqueous solution is pumped through the first tube by a first HPLC pump.

[0037] In some embodiments, the aqueous solution is pumped with a back pressure of at least 10 psi, 25 psi, 50 psi, 75 psi, or 100 psi.

[0038] In some embodiments, the first tube has an ID of 0.132 inches.

[0039] In some embodiments, the aqueous solution is pumped at a flow rate of at least 30 ml / min, 45 ml / min, 60 ml / min, 75 ml / min, 90 ml / min, 105 ml / min, 120 ml / min, 150 ml / min, 225 ml / min, 262.5 ml / min, 300 ml / min, or 450 ml / min.

[0040] In some embodiments, the aqueous solution containing RNA comprises about 2 mM to 50 mM citrate buffer at a pH of about 3.0 to 4.5.

[0041] In some embodiments, the aqueous solution containing RNA further comprises about 10 mM to 200 mM NaCl.

[0042] In some embodiments, the ethanol solution is pumped through a second tube by a second HPLC pump.

[0043] In some embodiments, the ethanol solution is pumped with a back pressure of at least 40 psi, 80 psi, 150 psi, 300 psi, or 400 psi.

[0044] In some embodiments, the second tube has an ID of 0.007 inches, 0.01 inches, or 0.02 inches.

[0045] In some embodiments, the ethanol solution is pumped at a flow rate of at least 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 50 ml / min, 60 ml / min, or 75 ml / min, 87.5 ml / min, 100 ml / min, or 150 ml / min.

[0046] In some embodiments, the first and second mixtures are maintained at 15-20°C.

[0047] In some embodiments, the mixing module comprises a second tube mounted perpendicular to a first tube, the first tube having an opening through its wall, the opening being approximately the size of the outer diameter of the second tube, and the second tube mating with the opening so that the second solution in the second tube can be transferred continuously to the first solution in the first tube.

[0048] In some embodiments, the mixing module is made of stainless steel tubing.

[0049] In some embodiments, the method further comprises pumping the dilution buffer through a third tube and introducing the dilution buffer into the discharge solution at the region of the Y connector, thereby mixing the dilution buffer with the discharge solution to produce a diluted discharge solution.

[0050] In some embodiments, the method further includes pumping the first dilution buffer through the third tube and introducing the first dilution buffer into the discharge solution at the region of the first Y connector, thereby mixing the first dilution buffer with the discharge solution to produce a first diluted discharge solution.

[0051] In some embodiments, the first dilution buffer comprises about 10 mM to 20 mM Tris buffer at a pH of about 7.4 to 8.5, about 45 mM to 55 mM NaCl, and about 8% to 10% sucrose.

[0052] In some embodiments, the first dilution buffer comprises about 10 mM to 20 mM Tris buffer at a pH of about 7.4 to 8.5, about 45 mM to 55 mM NaCl, and about 8% to 10% sucrose, and the RNA is siRNA.

[0053] In some embodiments, the method further includes pumping a second dilution buffer through a fourth tube and introducing the second dilution buffer into the first diluted effluent solution in the region of a second Y-connector, thereby mixing the second dilution buffer with the first diluted effluent solution to produce a second diluted effluent solution.

[0054] In some embodiments, the first dilution buffer comprises about 40 mM to 90 mM phosphate buffer at a pH of about 6.0 to 6.5; and the second dilution buffer comprises about 20 mM to 50 mM HEPES buffer at a pH of about 7.4 to 8.5, about 50 mM to 300 mM NaCl, and about 0% to 15% sucrose.

[0055] In some embodiments, the second dilution buffer comprises about 20 mM to 50 mM HEPES buffer at a pH of about 7.4 to 8.5, about 50 mM to 300 mM NaCl, and about 0% to 15% sucrose.

[0056] In some embodiments, the second dilution buffer further comprises about 25 mM to 100 mM NaCl.

[0057] In some embodiments, the first dilution buffer comprises about 40 mM to 90 mM phosphate buffer at a pH of about 6.0 to 6.5, and the RNA is mRNA.

[0058] In some embodiments, the first dilution buffer comprises about 40 mM to 90 mM phosphate buffer at a pH of about 6.0 to 6.5; and the second dilution buffer comprises about 20 mM to 50 mM HEPES buffer at a pH of about 7.4 to 8.5, about 50 mM to 300 mM NaCl, and about 0% to 15% sucrose, and the RNA is mRNA.

[0059] In some embodiments, the dilution buffer comprises about 5 to 25 mM Tris, 15 to 75 mM NaCl, and about 3 to 12% sucrose, pH about 7.0 to 8.5; about 5 to 20 mM Tris, about 20 to 70 mM NaCl, and about 3 to 12% sucrose, pH about 5.5 to 8.0, pH about 7.0 to 8.5; about 20 to 65 mM phosphate; about 10 to 30 mM HEPES, about 25 to 75 mM NaCl, and about 5 to 12% sucrose, pH about 7.0 to 8.5; or about 25 to 65 mM HEPES, about 25 to 65 mM NaCl, and about 3 to 12% sucrose, pH about 7.0 to 8.5. In some embodiments, the dilution buffer comprises 15 mM Tris, 50 mM NaCl, 9% sucrose at pH 7.5; 10 mM Tris, 50 mM NaCl, 9% sucrose at pH 7.5; 45 mM phosphate, pH 6.0 at about pH 7.4-8.0; 20 mM HEPES, 50 mM NaCl, 9% sucrose; or 50 mM HEPES, 50 mM NaCl, 9% sucrose at about pH 7.4-8.0. In some embodiments, the dilution buffer comprises 15 mM Tris, 50 mM NaCl, 9% sucrose at pH 7.5; 10 mM Tris, 50 mM NaCl, 9% sucrose at pH 7.5; 45 mM phosphate at pH 6.0; 20 mM HEPES, 50 mM NaCl, 9% sucrose at pH 7.4-8.0; or 50 mM HEPES, 50 mM NaCl, 9% sucrose at pH 7.4-8.0.

[0060] In some embodiments, the diluted effluent solution comprises 6.25% ethanol; 8.25% ethanol; 8.3% ethanol; or 12.5% ethanol.

[0061] In some embodiments, the Y connector is joined at an angle of about 45°.

[0062] In some embodiments, the Y connector is made of polyetheretherketone.

[0063] In some embodiments, the dilution buffer is pumped through the third tube at a flow rate of 400 to 900 mL / min.

[0064] In some embodiments, the third tube has an ID of 0.25 inches.

[0065] In some embodiments, the RNA encapsulated in the lipid nanoparticles is at least 70%, 75%, 80%, or 85% of the RNA mixed with the lipids.

[0066] In some embodiments, the lipid in the lipid-encapsulated RNA nanoparticles is at least 70%, 75%, 80%, or 85% of the lipid mixed with the RNA.

[0067] In some embodiments, X 7 is S.

[0068] In some embodiments, R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0069] In some embodiments, L 5 and L 6 are each independently C 1- C 10 In some embodiments, L 5 is C1-C3 alkyl, and L 6 is C 1- In some embodiments, L 6 is C 1- In some embodiments, L 5 and L 6 Each is a straight chain C7 alkyl. In some embodiments, L5 and L 6 are each a straight chain C9 alkyl.

[0070] In some embodiments, R 5 and R 6 are each independently alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C 2- In some embodiments, R 5 and R 6 Each alkenyl independently contains one double bond. 5 and R 6 Each is alkyl. In some embodiments, R 5 is a branched alkane. In some embodiments, R 5 and R 6 are each independently selected from the group consisting of C alkyl, C alkenyl, and C alkynyl. 5 and R 6 are each independently C 11 Alkyl, C 11 Alkenyl and C 11 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 are each independently selected from the group consisting of C alkyl, C alkenyl, and C alkynyl. 5 is -CH((CH2) p CH3)2 or -CH((CH2) p CH3)((CH2) p-1 CH3), where p is 4 to 8. In some embodiments, p is 5 and L 5 is C 1- In some embodiments, p is 6 and L 5 is C alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is C 1- In some embodiments, R 5 is -CH((CH2) p CH3)((CH2) p-1 CH3), where p is 7 or 8.

[0071] In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene.

[0072] In some embodiments, L 7 does not exist, R 4 is ethylene and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, R 4 is n-propylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, R 4 is ethylene and X 7 is S and R 7 and R 8 are each ethyl.

[0073] In some embodiments, the cationic lipid is [ka] [ka] [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0074] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0075] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0076] In some embodiments, the lipid is [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0077] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0078] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0079] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0080] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0081] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0082] In some embodiments, the cationic lipid is [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0083] In some embodiments, the lipid-encapsulated RNA nanoparticles have an average particle size of less than about 100 nm, hi some embodiments, the lipid-encapsulated RNA nanoparticles have an average particle size of about 55 nm to about 85 nm.

[0084] In some embodiments, the lipid-encapsulated RNA nanoparticles encapsulate at least about 50% of the RNA. In some embodiments, the lipid-encapsulated RNA nanoparticles encapsulate at least about 85% of the RNA.

[0085] In some embodiments, the lipid-encapsulated RNA nanoparticles further comprise a helper lipid selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC).In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC).

[0086] In some embodiments, the lipid-encapsulated RNA nanoparticles further comprise cholesterol.

[0087] In some embodiments, the lipid-encapsulated RNA nanoparticles further comprise a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG (dimyristoylglycerol).

[0088] In some embodiments, the lipid portion of the lipid-encapsulated RNA nanoparticles comprises about 48 mol% to about 66 mol% ionizable cationic lipid, about 2 mol% to about 12 mol% DSPC, about 25 mol% to about 42 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG. In some embodiments, the lipid portion of the lipid-encapsulated RNA nanoparticles comprises about 50 mol% to about 61 mol% ionizable cationic lipid, about 5 mol% to about 9 mol% DSPC, about 29 mol% to about 38 mol% cholesterol, and about 1 mol% to about 2 mol% PEG2000-DMG. In some embodiments, the lipid portion of the lipid-encapsulated RNA nanoparticles comprises about 56 mol% to about 58 mol% ionizable cationic lipid, about 6 mol% to about 8 mol% DSPC, about 31 mol% to about 34 mol% cholesterol, and about 1.25 mol% to about 1.75 mol% PEG2000-DMG.

[0089] In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 50:1 to about 3:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 50:1 to about 5:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 50:1 to about 10:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 40:1 to about 20:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 35:1 to about 25:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 28:1 to about 32:1. In some embodiments, the weight ratio of total lipid:RNA in the lipid-encapsulated RNA nanoparticles is about 29:1 to about 31:1.

[0090] In some embodiments, the lipid-encapsulated RNA nanoparticles have a cationic lipid:DOTAP:DSPC:cholesterol:PEG ratio of 25:25:10:38.5:1.5, 25:25:10:37:3, 25:25:10:35:5, 20:20:7:51.5:1.5, 25:20:10:42:3, 20:30:13:32:5, 25:20:10:40:5, 25:25:13:35.5:1.5 , 25:30:7:35:3, 30:20:13:34:3, 30:25:7:33:3, 30:30:10:25.8:1.5, 15:20:13:49:3, 20:20:13:44:3, 20:25:13:39:3, 15:25:13:44:3, 20:25:13:39:3, 25:25:13:34:3, 30:20:13:34:3 or 30:30:13:29:3.

[0091] In some embodiments, the lipid-encapsulated RNA nanoparticles comprise about 20% to 60% cationic lipids (w / w), hi some embodiments, the lipid-encapsulated RNA nanoparticles comprise less than about 90% cationic lipids (w / w).

[0092] In some embodiments, the lipid-encapsulated RNA nanoparticles comprise about 5% to 30% w / w of a helper lipid.

[0093] In some embodiments, the lipid-encapsulated RNA nanoparticles contain between about 0% and 60% w / w cholesterol.

[0094] In some embodiments, the lipid-encapsulated RNA nanoparticles comprise about 0.5% to 15% w / w of polyethylene glycol (PEG).

[0095] In some embodiments, the lipid-encapsulated RNA nanoparticles comprise about 5% to 25% w / w of neutral lipids.

[0096] In some embodiments, the lipid-encapsulated RNA nanoparticles comprise between about 0% and 30% w / w of phospholipids.

[0097] In some embodiments, the cholesterol:RNA molar ratio of the lipid-encapsulated RNA nanoparticles is between about 1.5:1 and 9:1.

[0098] In some embodiments, the lipid-encapsulated RNA nanoparticles have a molar ratio of PEG:mRNA of about 0.5:1 to 5:1.

[0099] In some embodiments, the lipid-encapsulated RNA nanoparticles have a molar ratio of helper lipid:RNA of about 0.25:1 to 4:1.

[0100] In some embodiments, the molar ratio of cationic lipid:RNA of the lipid-encapsulated RNA nanoparticles is about 1:1 to 7:1. In some embodiments, the lipid-encapsulated RNA nanoparticles comprise about 10 wt% to 98 wt% of a cationic lipid represented by Formula I. In some embodiments, the molar ratio of cationic lipid:RNA of the lipid-encapsulated RNA nanoparticles is about 5.4:1 to 15.4:1. In some embodiments, the molar % of cationic lipid and / or the cationic lipid:RNA ratio of the lipid-encapsulated RNA nanoparticles are as shown in Figure 2. In some embodiments, the cationic lipid composition and / or the molar % of lipid of the lipid-encapsulated RNA nanoparticles are as shown in Figure 3.

[0101] In some embodiments, the RNA is selected from the group consisting of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), and self-replicating RNA.

[0102] In some embodiments, the average particle size is less than 70 nm, 80 nm, 90 nm, or 100 nm.

[0103] In some embodiments, the polydispersity index is less than 0.05, 0.07, or 0.09.

[0104] In some embodiments, RNA encapsulation is greater than 90%, 94%, 96% or 98%.

[0105] In some embodiments, the batch size is 0.05 to 100 g RNA, In some embodiments, the batch size is 0.05 to 30 g RNA.

[0106] In some embodiments, the variation in average particle size between batches is less than 10%.

[0107] Natural and modified nucleotides Preferably, the mRNA described herein contains one or more chemically modified nucleotides. Examples of nucleic acid monomers include non-natural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides may be artificially modified at either the base or sugar moiety. In nature, most polynucleotides contain nucleotides that are "unmodified" or "natural" nucleotides, including the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). These bases are typically fixed to ribose or deoxyribose at the 1' position. The use of mRNA polynucleotides containing chemically modified nucleotides has been shown to improve mRNA expression, expression rate, half-life, and / or expressed protein concentration. mRNA polynucleotides containing chemically modified nucleotides are also useful for optimizing protein localization, thereby avoiding adverse biological responses, such as immune responses and / or degradation pathways.

[0108] Examples of modified or chemically modified nucleotides are 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N4-alkylcytidine, N 4 -aminocytidine, N 4 -acetylcytidine and N 4 ,N 4 -Contains dialkylcytidine.

[0109] Examples of modified or chemically modified nucleotides are 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; N 4 -methylcytidine, N 4 -aminocytidine, N 4 -acetylcytidine and N 4 ,N 4-Contains dimethylcytidine.

[0110] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine and 6-alkyluridine.

[0111] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

[0112] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'-O-dimethyluridine.

[0113] Examples of modified or chemically modified nucleotides are N 6 -methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -methyladenosine, N 6-Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyl-adenosine, N 6 -methyl-N 6 -Threonylcarbamoyl-adenosine, 2-methylthio-N 6 -Threonylcarbamoyl-adenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyladenosine, 2-methylthio-N 6 -hydroxynorvalylcarbamoyl-adenosine, N 6 -acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-O-methyl-adenosine, N 6 ,2'-O-dimethyl-adenosine,N 6 ,N 6 ,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N 6 -methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N 6 -(19-amino-pentaoxanonadecyl)-adenosine.

[0114] Examples of modified or chemically modified nucleotides are N l -Alkylguanosine, N 2 -Alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-alkylguanosine, xanthosine, inosine and N l -Contains alkylinosines.

[0115] Examples of modified or chemically modified nucleotides are N l -methylguanosine, N 2 -methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-methylguanosine, xanthosine, inosine and N l -Contains methylinosine.

[0116] An example of a modified or chemically modified nucleotide is pseudouridine. l -Alkylpseudouridine, N l -Cycloalkylpseudouridine, N 1 -Hydroxypseudouridine, N 1 -Hydroxyalkylpseudouridine, N l -phenylpseudouridine, N l -phenylalkylpseudouridine, N l -aminoalkylpseudouridine, N 3 -Alkylpseudouridine, N 6 -Alkylpseudouridine, N 6 -Alkoxypseudouridine, N 6 -Hydroxypseudouridine, N 6 -Hydroxyalkylpseudouridine, N 6 -Morpholinopseudouridine, N 6 -phenylpseudouridine and N 6 -halopseudouridines. Examples of pseudouridines include N l -Alkyl-N 6 -Alkylpseudouridine, N l -Alkyl-N 6 -Alkoxypseudouridine, N l -Alkyl-N 6 -Hydroxypseudouridine, N l -Alkyl-N 6 -Hydroxyalkylpseudouridine, N l -Alkyl-N 6 -Morpholinopseudouridine, N l -Alkyl-N 6 -phenylpseudouridine and Nl -Alkyl-N 6 In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

[0117] An example of pseudouridine is N l -methylpseudouridine (also referred to herein as "N1MPU"), N l -Ethylpseudouridine, N l -Propylpseudouridine, N l -Cyclopropylpseudouridine, N l -phenylpseudouridine, Nl-aminomethylpseudouridine, N 3 -methylpseudouridine, N 1 -hydroxypseudouridine and N 1 -Contains hydroxymethylpseudouridine.

[0118] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0119] Examples of modified and chemically modified nucleotide monomers include any such nucleotide known in the art, such as 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxy abasic monomer residues.

[0120] Examples of modified and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides and 3'-inverted thymidines.

[0121] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoronucleotides, and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0122] Examples of modified and chemically modified nucleotide monomers include 2',4'-tethered 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0123] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides and 2'-O-allyl nucleotides.

[0124] Examples of modified and chemically modified nucleotide monomers are N 6 -Contains methyl adenosine nucleotides.

[0125] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0126] Examples of modified and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0127] Examples of modified and chemically modified nucleotide monomers include replacement of the 2'-OH group of the nucleotide with 2'-R, 2'-OR, 2'-halogen, 2'-SR, or 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0128] The above examples of base modifications can be combined with further modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers can be found in nature.

[0129] The preferred nucleotide modification is N 1 -methylpseudouridine and 5-methoxyuridine.

[0130] 5' Cap The cap structure at the 5' end of mRNA (or self-replicating RNA), present in all eukaryotes (and some viruses), is important for stabilizing mRNA in vivo. The naturally occurring cap structure contains a riboguanosine residue that is methylated at position N7 of the guanine base. This 7-methylguanosine (m 7 G) binds to the mRNA molecule via the 5'-to-5'-triphosphate chain at the 5' end. The presence of the m7Gppp fragment at the 5' end is essential for mRNA maturation because it protects mRNA from exonucleolytic degradation, promotes mRNA transport from the nucleus to the cytoplasm, and plays an important role in the assembly of the translation initiation complex (Cell 9:645-653, (1976); Nature 266:235, (1977); Federation of Experimental Biologists Society Letter 96:1-11, (1978); Cell 40:223-24, (1985); Prog. Nuc. Acid Res. 35:173-207, (1988); Ann. Rev. Biochem. 68:913-963, (1999); J. Biol. Chem. 274:30337-3040, (1999)).

[0131] Only capped mRNAs are active in cap-dependent translation; "truncating" an mRNA results in almost complete loss of template activity for protein synthesis (Nature, 255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); Proc. Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

[0132] Another component of eukaryotic mRNA is the presence of 2'-O-methylnucleoside residues at position 1 of the transcript (cap 1) and, in some cases, at positions 1 and 2 of the transcript (cap 2). 2'-O-methylation of mRNA provides higher efficiency of mRNA translation in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves the nuclease stability of 5'-capped mRNA. mRNAs with cap 1 (and cap 2) are distinguishing marks that allow cells to recognize authentic mRNA 5' ends and, in some cases, distinguish transcripts emanating from infectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).

[0133] Some examples of 5' cap structures and methods for making them are shown in WO2015 / 051169A2, WO2015 / 061491, US2018 / 0273576, and U.S. Patent Nos. 8,093,367, 8,304,529, and 10,487,105. In some embodiments, the 5' cap is a 5' cap, as known in the art. 7 In some embodiments, the 5' cap is GpppAmpG. 7 GpppG or m 7 GpppGmd. The structural formula of an embodiment of the 5' cap structure is shown below:

[0134] In some embodiments, the mRNA described herein has the formula (Cap I): [ka] (Cap I) [In the formula, B 1 is a natural or modified nucleobase; R 1 and R 2 are each independently selected from halogen, OH, and OCH; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; n is 0, 1, 2, or 3; and mRNA represents an mRNA linked at its 5' end. In some embodiments, G is guanine, and in some embodiments, n is 0. In some embodiments, B 1 is A or m6A, and R 1 is OCH3; G is guanine; m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0135] In some embodiments, the mRNA described herein has the formula (Cap II): [ka] (Cap II) [In the formula, B 1 and B 2 are each independently a natural or modified nucleobase; R 1 , R 2 and R 3 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 3 at least one of which is OH] In some embodiments, G is guanine, and in some embodiments, n is 0. In some embodiments, B 1 is A or m6A, and R 1 is OCH3; G is guanine; m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0136] In some embodiments, the mRNA described herein has the formula (Cap III): [ka] (Cap III) [In the formula, B 1 , B 2 and B 3 are each independently a natural or modified nucleobase; R 1 , R 2 , R 3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] In some embodiments, G is a guanine, and in some embodiments, B 1 is A or m6A, and R 1 is OCH3; G is guanine; m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6In some embodiments, n is 1.

[0137] In some embodiments, the mRNA described herein has the formula (Cap IV): [ka] (Cap IV) [In the formula, R 1 , R 2 and R 3 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 3 at least one of which is OH] m having the structure 7 In some embodiments, the 5' cap comprises a GpppG 5' cap analog. 7 GpppG, where R 1 , R 2 and R 3 are each OH, n is 1, and L 1 is phosphate. In some embodiments, n is 1. In some embodiments, R 1 and R 2 are each OH and R 3 is OCH3, each P is a phosphate, thereby forming a phosphodiester bond, and the mRNA is an mRNA of the present disclosure attached at its 5' end, n is 1, and L 1 is a phosphate.

[0138] In some embodiments, the mRNA described herein has the formula (Cap V): [ka] (Cap V) [In the formula, R 1 , R 2 and R 3 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 3 at least one of which is OH] m having the structure 7 Gpppm 7 In some embodiments, n is 0.

[0139] In some embodiments, the mRNA described herein is 7 Gpppm 7 GpN, including 5' cap analogs, where N is a natural or modified nucleotide, and the 5' cap analog has the formula (Cap VI): [ka] (Cap VI) [In the formula, B 3 is a natural or modified nucleobase; R 1 , R 2 , R 3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] In some embodiments, G is guanine, and in some embodiments, B 1 is A or m6A, and R 1 is OCH3; G is guanine; m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 In some embodiments, n is 1.

[0140] In some embodiments, the mRNA described herein has the structure of formula (Cap VII): [ka] (Cap VII) [In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] m having the structure 7 Gpppm 7 In some embodiments, n is 1.

[0141] In some embodiments, the mRNA described herein has the formula (Cap VIII): [ka] (Cap VIII) [In the formula, R 1 , R 2 , R3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] m having the structure 7 Gpppm 7 Gpm 7 In some embodiments, n is 1.

[0142] In some embodiments, the mRNA described herein has the formula (Cap IX): [ka] (Cap IX) [In the formula, R 1 , R 2 and R 3 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 3 at least one of which is OH] m having the structure 7 In some embodiments, n is 1.

[0143] In some embodiments, the mRNA described herein is 7GpppApN 5' cap analogs, wherein N is a natural or modified nucleotide and the 5' cap has the formula (Cap X) [ka] (Cap X) [In the formula, B 3 is a natural or modified nucleobase; R 1 , R 2 , R 3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] In some embodiments, B 3 , G, m 7 G, A or m 6 A is guanine; G is guanine; m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 In some embodiments, n is 1.

[0144] In some embodiments, the mRNA described herein has the formula (Cap XI): [ka] (Cap XI) [In the formula, R 1 , R 2 and R 4are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 4 at least one of which is OH] m having the structure 7 In some embodiments, the compound of formula XI comprises a 5' cap analog of GpppAmpG. 7 GpppAmpG, where R 1 , R 2 and R 4 are each OH, n is 1, and L 1 is a phosphate bond. In some embodiments, n is 1.

[0145] In some embodiments, the mRNA described herein has the structure of formula (Cap XII): [ka] (Cap XII) [In the formula, R 1 , R 2 , R 3 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 , R 3 and R 4 at least one of which is OH] m having the structure 7 GpppApm 7In some embodiments, n is 1.

[0146] In some embodiments, the mRNA described herein has the structure of formula (Cap XIII): [ka] (Cap XIII) [In the formula, R 1 , R 2 and R 4 are each independently selected from halogen, OH, and OCH3; each P is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate; mRNA represents an mRNA linked at its 5' end; n is 0, 1, 2, or 3; L 1 is a phosphate, phosphorothioate, or boranophosphate, and R 1 , R 2 and R 4 at least one of which is OH] m having the structure 7 GpppAmpm 7 In some embodiments, n is 1.

[0147] The RNA may include a 5' trinucleotide cap structure as described in US2018 / 0105551A1, which is incorporated herein by reference in its entirety.

[0148] Naturally occurring RNA can have a phosphate backbone. The RNA described herein can contain other types of backbones and bases, including peptide nucleic acids, phosphothionate, phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0149] cationic lipids The lipid formulation preferably contains a cationic lipid suitable for forming cationic liposomes or lipid nanoparticles. Cationic lipids have been widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails or steroid moieties, and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at approximately physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / short hairpin RNA-shRNA. Cationic lipids, such as DOTAP (l,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate), form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, providing high in vitro transfection efficiency.

[0150] In the lipid formulations and methods of making the same of the present disclosure, the cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride), N-(1-(2,3- Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DA) P), l,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl) ), l,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-diethyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodeca DLin-2-ol (C12-200), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3l-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-l-amine (MC4 Ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), l,2-dileoyl-sn-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethylammonium trifluoroacetate (DOSPA), and N-(l-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium trifluoroacetate (DOSPA).Cationic lipids include, but are not limited to, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). Commercially available preparations of cationic lipids may also be used, such as, for example, LIPOFECTIN (containing DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (containing DOSPA and DOPE, available from GIBCO / BRL).

[0151] Other suitable cationic lipids are disclosed in WO09 / 086558, WO09 / 127060, WO10 / 048536, WO10 / 054406, WO10 / 088537, WO10 / 129709 and WO2011 / 153493; U.S. Patent Application Publication Nos. 2011 / 0256175, 2012 / 0128760 and 2012 / 0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0152] Other suitable cationic lipids include those with different fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids called amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. Generally, amino lipids with fewer saturated acyl chains are easier to size, especially when the size of the complexes needs to be less than about 0.3 μm for sterilization by filtration. Amino lipids containing unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. Other scaffolds can be used to separate the amino group from the fatty acid or fatty alkyl portion of the amino lipid.

[0153] In some embodiments, the lipid formulations and methods for making same comprise a cationic lipid having Formula I according to patent application PCT / EP2017 / 064066, the disclosure of which is also incorporated herein by reference.

[0154] In some embodiments, the amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group ("ionizable cationic lipid"), such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood, of course, that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species and does not require that all lipids exist in a charged or neutral form. Lipids that have one or more protonatable or deprotonatable groups or are zwitterionic are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group ranging from about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionizable cationic lipid is about 6 to about 7.

[0155] In some embodiments, the lipid formulations and methods for making same have Formula I: [ka] (I) [In the formula, R 5 and R 6 are each independently a straight-chain or branched-chain C 1- C 31 Alkyl, C 2- C 31 Alkenyl or C 2- C 31 selected from the group consisting of alkynyl and cholesteryl; L 5 and L 6are each independently a linear C 1- C 20 Alkyl and C 2- C 20 alkenyl; X 5 is -C(O)O-, which results in -C(O)OR 6 is formed or -OC(O)-, whereby -OC(O)-R 6 is formed; X 6 is -C(O)O-, which results in -C(O)OR 5 is formed or -OC(O)-, whereby -OC(O)-R 5 is formed; X 7 is S or O; L 7 is absent or lower alkyl; R 4 is a linear or branched chain C 1- C alkyl; and R 7 and R 8 are each independently hydrogen and a straight or branched chain C 1- C6 alkyl] or a pharmaceutically acceptable salt or solvate thereof.

[0156] In some embodiments, X 7 is S.

[0157] In some embodiments, X 5 is -C(O)O-, which results in -C(O)OR 6 is formed, and X 6 is -C(O)O-, which results in -C(O)OR 5 is formed.

[0158] In some embodiments, R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0159] In some embodiments, L 5 and L6 are each independently C 1- C 10 In some embodiments, L 5 is C1-C3 alkyl, and L 6 is C 1- In some embodiments, L 6 is C 1- In some embodiments, L 5 and L 6 Each is a straight chain C7 alkyl. In some embodiments, L 5 and L 6 are each a straight chain C9 alkyl.

[0160] In some embodiments, R 5 and R 6 are each independently alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C 2- C alkenyl. In some embodiments, the alkenyl contains one double bond. In some embodiments, R 5 and R 6 Each is alkyl. In some embodiments, R 5 is branched alkyl. In some embodiments, R 5 and R 6 are each independently selected from the group consisting of C alkyl, C alkenyl, and C alkynyl. 5 and R 6 are each independently C 11 Alkyl, C 11 Alkenyl and C 11 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 are each independently selected from the group consisting of C alkyl, C alkenyl, and C alkynyl. 5 is -CH((CH2)p CH3)2 or -CH((CH2) p CH3)((CH2) p-1 CH3), where p is 4 to 8. In some embodiments, p is 5 and L 5 is C 1- In some embodiments, p is 6 and L 5 is C alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L 5 is C 1- In some embodiments, R 5 is -CH((CH2) p CH3)((CH2) p-1 CH3), where p is 7 or 8.

[0161] In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene.

[0162] In some embodiments, L 7 does not exist, R 4 is ethylene and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, R 4 is n-propylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, R 4 is ethylene and X 7 is S and R 7 and R 8 are each ethyl.

[0163] In some embodiments, X 7 is S and X5 is -C(O)O-, which results in -C(O)OR 6 is formed, and X 6 is -C(O)O-, which results in -C(O)OR 5 is formed, and L 5 and L 6 are each independently a linear C 3- C7 alkyl, L 7 does not exist, R 5 is -CH((CH2) p CH3)2 and R 6 is C 7- C 12 In some preferred embodiments, p is 6 and R 6 is a C9 alkenyl.

[0164] In some embodiments, the lipid formulations and methods for producing the same comprise: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The ionizable cationic lipid comprises an ionizable cationic lipid selected from the group consisting of:

[0165] In some embodiments, the lipid formulation may comprise an ionizable cationic lipid selected from the group consisting of lipid #1 through lipid #9 shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3]

[0166] In some embodiments, the lipid formulation comprises: [ka] or a pharmaceutically acceptable salt thereof.

[0167] In some preferred embodiments, the lipid formulation comprises: [ka] or a pharmaceutically acceptable salt thereof.

[0168] In one embodiment, any one or more lipids described herein may be explicitly excluded.

[0169] Helper lipids and sterols The mRNA-lipid formulations and methods for producing them of the present disclosure can include helper lipids, which can be referred to as neutral helper lipids, non-cationic lipids, non-cationic helper lipids, anionic lipids, anionic helper lipids or neutral lipids.Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to increase cellular uptake when helper lipids are present in the formulation (Curr. Drug Metab. 2014; 15(9):882-92). For example, several studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), which are more fusogenic (i.e., promote fusion) than cationic lipids, affect the polymorphic characteristics of lipid-nucleic acid complexes, promoting the transition from lamellar to hexagonal phases and thereby inducing cell membrane fusion and disruption (Nanomedicine (Lond). 2014 Jan;9(1):105-20). Additionally, the use of helper lipids can help reduce the potential adverse effects of many common cationic lipids, such as toxicity and immunogenicity.

[0170] Non-limiting examples of non-cationic lipids suitable for the lipid formulations and methods of making the same disclosed herein include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (PAPC ... Examples of suitable phospholipids include mitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably C 10 -C 24 It is an acyl group derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0171] Further examples of non-cationic lipids include sterols, such as cholesterol and its derivatives. One study concluded that cholesterol, as a helper lipid, increases the charge spacing of the lipid layer in contact with nucleic acid, making the charge distribution more closely match that of nucleic acid (JR Soc. Interface. 2012 Mar 7; 9(68): 548-561). Non-limiting examples of cholesterol derivatives include polar analogs, such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs, such as 5α-cholestan, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether.

[0172] In some embodiments, the helper lipid present in lipid formulation comprises or consists of the mixture of one or more phospholipids and cholesterol or its derivatives.In other embodiments, the neutral lipid present in lipid formulation comprises or consists of one or more phospholipids, for example, cholesterol-free lipid formulations.In still other embodiments, the neutral lipid present in lipid formulation comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid formulations.

[0173] Other examples of helper lipids include non-phosphorus-containing lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol retinol, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0174] In some embodiments, the helper lipid comprises about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation.

[0175] The cholesterol or cholesterol derivative in the lipid formulations and methods for producing same may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 35 mol%, or about 28 mol% to about 35 mol% of the total lipid present in the lipid formulation; or about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, or about 37 mol%.

[0176] In some embodiments, the phosphorus component in the mixture comprises about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation.

[0177] The percentage of helper lipid present in the lipid formulation and the method for producing it is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, ±5 mole %.

[0178] Lipid formulations containing cationic lipid compounds or ionizable cationic lipid compounds may contain, on a molar basis, about 30-70% cationic lipid compounds, about 25-40% cholesterol, about 2-15% helper lipids, and about 0.5-5% polyethylene glycol (PEG) lipids, which are the percentage of the total lipids present in the formulation. In some embodiments, the composition is about 40-65% cationic lipid compounds, about 25-35% cholesterol, about 3-9% helper lipids, and about 0.5-3% PEG-lipids, which are the percentage of the total lipids present in the formulation.

[0179] The formulation may be a lipid particle formulation comprising, for example, 8-30% nucleic acid compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipid, or even 100% cationic lipid.

[0180] lipid conjugates The lipid-encapsulated RNA nanoparticles described herein can further comprise lipid conjugates.Conjugated lipids are useful in preventing particle aggregation.Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.In addition, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the end of the attached PEG chain (Front Pharmacol. 2015 Dec 1; 6:286).

[0181] In a preferred embodiment, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) as a coating or surface ligand in lipid formulations, a technique known as PEGylation, helps protect nanoparticles from the immune system and evade uptake by the reticuloendothelial system (RES) (Nanomedicine (Lond). 2011 Jun; 6(4):715-28). PEGylation is widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Surfactant-like PEG-lipids (e.g., PEG-DSPE) can enter the lipid formulation and form a hydration layer and a steric barrier on the surface. Based on the degree of PEGylation, the surface layer can generally be divided into two types: a brush-like layer and a mushroom-like layer. In formulations stabilized with PEG-DSPE, PEG adopts a mushroom-like conformation at low PEGylation levels (usually less than 5 mol%) and transitions to a brush-like conformation when the PEG-DSPE content exceeds a certain level (Journal of Nanomaterials. 2011; 2011:12). Increasing PEGylation has been shown to result in a significant increase in the circulating half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13():507-30; J. Control Release. 2010 Aug 3; 145(3):178-81).

[0182] Suitable examples of PEG lipids include PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide, PEG conjugated to cholesterol or derivatives thereof, and mixtures thereof.

[0183] PEG is a linear, water-soluble polymer of repeating ethylene PEG units with two terminal hydroxyl groups. PEGs are classified by molecular weight and include monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), and compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0184] The PEG moiety of the PEG-lipid conjugates described herein can comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In particular examples, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight can be any value or subvalue within the stated range, including the endpoints.

[0185] In certain examples, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl group.PEG can be directly conjugated with lipid, or can be linked with lipid via linker moiety.For example, any linker moiety suitable for connecting PEG with lipid can be used, including non-ester-containing linker moiety and ester-containing linker moiety.In a preferred embodiment, linker moiety is non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCHCHC(O)-), succinamidyl (-NHC(O)CHCHC(O)NH-), ether, and combinations thereof (e.g., linkers containing both carbamate and amide linker moieties). In a preferred embodiment, a carbamate linker is used to attach PEG to the lipid.

[0186] In other embodiments, PEG is attached to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0187] Phosphatidylethanolamines with various acyl chain lengths and degrees of saturation can be conjugated with PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20 are preferred. Phosphatidylethanolamines containing mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0188] In some embodiments, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate or PEG-distearyloxypropyl (C 18 ) conjugates. In these embodiments, the PEG preferably has an average molecular weight of about 750 or about 2,000 daltons. In certain embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0189] In addition, other hydrophilic polymers can be used instead of PEG.Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0190] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein). In other embodiments, lipid conjugate (for example, PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5% (or any fraction thereof or within any range) of the total lipid present in lipid formulation and its manufacturing method.Amount can be any value or subvalue within the stated range, including endpoints.

[0191] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations and methods for producing the same of the present disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation can vary, for example, by ±0.5 mol%. Those skilled in the art will understand that the concentration of lipid conjugate can vary depending on the lipid conjugate used and the rate at which the lipid formulation becomes fusogenic.

[0192] Formation of lipid-encapsulated RNA nanoparticles FIG. 1 is an example of a representative flow chart of the method described herein for producing lipid-encapsulated RNA nanoparticles.

[0193] The mixing bed configuration consists of a first tube for transporting the aqueous solution having an internal diameter (ID) greater than 0.1 inches, preferably greater than 0.132 inches; a second tube for transporting the ethanol solution having an ID greater than 0.005 inches, preferably greater than 0.01 inches, where the second (organic) tube intersects the first (aqueous) tube at or near a perpendicular angle. The discharge flow rate during mixing is at least 200 ml / min, preferably at least 300 ml / min.

[0194] The method described herein provides, for example, an aqueous RNA solution containing therapeutic RNA, which is prepared under Good Manufacturing Practice (GMP) and solubilized in a buffer solution, for example, an aqueous solution containing citric acid.The method of the present invention also provides an organic solution containing one or more lipids, for example, clinical-grade lipids, which are synthesized under GMP and produced by solubilizing lipids in a water-miscible organic solvent.In the method described herein, the water-miscible organic solvent is preferably a lower alkanol, for example, ethanol.Preferably, both solutions are filter sterilized, and their concentrations are adjusted.

[0195] The organic lipid solution is mixed with an aqueous solution containing nucleic acid to form lipid-encapsulated RNA nanoparticles having a lamellar morphology, i.e., comprising a lipid bilayer. In one embodiment, the nucleic acid is encapsulated in the lipid-encapsulated RNA nanoparticles, forming a lamellar structure.

[0196] The method described herein involves continuously introducing a lipid solution into an aqueous solution in a mixing environment, preferably vertically in a mixing module. The mixing dilutes the lipid solution with the aqueous solution to 20%, 22.5%, 25%, 27.5%, or 30% ethanol, preferably 25% ethanol, and forms lipid-encapsulated RNA nanoparticles in a turbulent flow.

[0197] After formation of lipid-encapsulated RNA nanoparticles, the mixture is serially diluted with buffer to 7.5%, 10%, 12.5%, or 15%, preferably less than 12.5%, ethanol, which further stabilizes the lipid-encapsulated RNA nanoparticles and increases the encapsulation of nucleic acids.

[0198] The lipid-encapsulated RNA nanoparticles are concentrated by tangential flow filtration, preferably using a hollow fiber filter. The concentrated lipid-encapsulated RNA nanoparticles are subjected to an ultrafiltration step to remove the alkanol and replace it with a buffer solution. The nucleic acid concentration is adjusted by dilution. The resulting formulation is filter-sterilized and filled into vials. This process is described in further detail below using the steps shown in Figure 1.

[0199] Lipid solubilization and RNA dissolution In one embodiment, the lipid-encapsulated RNA nanoparticles produced by the methods described herein are in the form of multimolecular assemblies of RNA and lipids, in which the RNA is at least partially encapsulated by ion pairing with cationic lipids.

[0200] The preferred size of lipid-encapsulated nanoparticles containing RNA produced by the methods described herein is about 50 to 200 nm in diameter, with a size distribution preferably having an average size (e.g., diameter) of about 70 nm to about 150 nm, and more preferably an average size of less than about 100 nm.

[0201] In certain embodiments, the lipid nanoparticles described herein comprise four components: a helper lipid; cholesterol; a PEG-lipid; and an ionizable cationic lipid. Preferably, the helper lipid is DSPC, the PEG-lipid is PEG-DMG, and the ionizable cationic lipid is an ionizable cationic lipid. In certain embodiments, the organic solvent concentration at which the lipid is solubilized is about 45% v / v to about 90% v / v. In certain preferred embodiments, the organic solvent is a lower alkanol. Suitable lower alkanols include, for example, methanol, ethanol, propanol, butanol, pentanol, their isomers, and combinations thereof. The solvent is preferably ethanol at a concentration of about 50% v / v to about 90% v / v. Preferably, the lipid occupies a volume of about 1 mL / g to about 5 mL / g.

[0202] Lipid is solubilized, for example, by using an overhead stirrer at suitable temperature.In one embodiment, the total lipid concentration of solution is about 49.4mg / mL.In certain preferred embodiments, RNA is contained in aqueous solution (for example, buffer solution) and diluted to final concentration.Preferably, final concentration is about 0.55mg / mL in citrate buffer solution, and pH is about 3.5.

[0203] RNA is preferably double-stranded RNA (dsRNA), mRNA or self-replicating RNA.The size of dsRNA is between 10 base pairs and several hundred bases, preferably less than 30 base pairs, most preferably less than 25 base pairs.The size of mRNA is single-stranded and is 10 to several thousand bases.

[0204] Formation steps of lipid-encapsulated RNA nanoparticles After preparing the organic solution and the aqueous solution, they are mixed together using the device described in detail below. Briefly, the device consists of a first tube for transporting the aqueous RNA solution and a second tube for transporting the organic lipid solution, where the second tube intersects perpendicularly with the first tube in a mixing module. The two solutions are pumped through their respective tubes by separate HPLC pumps and mixed vertically in the region of the first tube in the mixing module. Preferably, the aqueous RNA solution is pumped at a rate 2.0 times, 2.5 times, 3.0 times, 3.25 times, or 3.5 times, preferably 3.0 times, faster than the organic lipid solution. When the two solutions are mixed in the mixing region, lipid-encapsulated RNA nanoparticles are formed.

[0205] The pump speed and size of the first tube in the region of the mixing module provide a turbulent mixing method. In fluid mechanics, turbulence (or turbulent flow) is fluid motion characterized by chaotic changes in pressure and velocity. This contrasts with laminar flow, which occurs when fluids flow in parallel layers without disturbance between them. Turbulent flow is always highly irregular, and the readily available energy supply in turbulent flow tends to accelerate the homogenization (mixing) of fluid mixtures. The property responsible for enhanced mixing and increased rates of mass, momentum, and energy transfer within a flow is called "diffusivity." Other properties of turbulent flow include "rotationality," due to the powerful three-dimensional vortex generation mechanism known as vortex stretching, and "dissipation," due to the rapid dissipation of turbulent flow as kinetic energy is converted to internal energy by viscous shear stresses. Turbulent mixing is governed by the small-scale (compared to the parent flow) random motion of parcels within a fluid, which can bring parcels closer or further apart and allow them to intermix. The manufacturing methods described herein for mixing lipid and aqueous solutions provide for encapsulation of RNA into lipid nanoparticles formed upon their formation with encapsulation efficiencies of greater than 95%.

[0206] Lipid nanoparticles are typically formed at room temperature, but according to the present disclosure, lipid nanoparticles can be formed at high temperatures.There is no general requirement for the buffer composition.In fact, the manufacturing method and device of the present disclosure can produce lipid vesicles by mixing lipids in ethanol with RNA in aqueous solution.

[0207] In one embodiment, lipid nanoparticles are formed when lipids dissolved in an organic solvent, such as ethanol, are gradually diluted by mixing with an aqueous buffer solution, first by mixing the aqueous and lipid streams together in a mixing module to a final concentration, preferably 25-40%, of the lipid, solvent, and solute. The resulting lipid, solvent, and solute concentrations can be kept constant throughout the vesicle formation process. Following initial formation, the initial lipid-RNA mixture is further diluted by the addition of buffer, preferably to about 6.0%, 6.25%, 7.0%, 7.5%, 10%, 12.5%, or 15% organic solvent, most preferably 6.25%, 6.25%, 7.0%, 7.5%, 10%, or 12.5%.

[0208] The continuous method described herein is fully scalable. In one embodiment, lipid-encapsulated RNA nanoparticles are formed with an average diameter of less than about 80 nm without mechanical energy processes such as membrane extrusion, sonication, or microfluidization.

[0209] Lipid-encapsulated RNA nanoparticles The lipid-encapsulated RNA nanoparticles described herein comprise a nanoparticle or bilayer of lipid molecules. In addition to a cationic lipid (eg, an ionizable cationic lipid), the lipid-encapsulated RNA nanoparticles include a neutral lipid or a polymer.

[0210] In some embodiments, the RNA is completely encapsulated within the lipid portion of the lipid nanoparticle, resulting in the RNA in the lipid-encapsulated RNA nanoparticles being resistant to nuclease degradation in aqueous solution. In other embodiments, the lipid-encapsulated RNA nanoparticles described herein are substantially non-toxic to mammals, such as humans. The lipid-encapsulated RNA nanoparticles typically have an average diameter of 30 nm to 150 nm, 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, or 70 nm to 90 nm. The lipid-encapsulated RNA nanoparticles described herein also typically have a lipid:RNA ratio (mass / mass) of 1:1 to 100:1, 1:1 to 50:1, 2:1 to 25:1, 3:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 14:1, or 9:1 to 20:1. In some embodiments, the weight ratio of total lipids to RNA of the composition is about 50:1 to 10:1. In some embodiments, the weight ratio of total lipids to RNA of the composition is about 40:1 to 20:1. In some embodiments, the weight ratio of total lipids to RNA of the composition is about 35:1 to 25:1. In some embodiments, the weight ratio of total lipids to RNA of the composition is about 28:1 to 32:1.

[0211] In a preferred embodiment, lipid particles comprise RNA, cationic lipid (for example, one or more cationic lipids or their salts as described herein), phospholipid and conjugated lipid (for example, one or more PEG-lipid conjugates) that prevent particle aggregation.Lipid-encapsulated RNA nanoparticles can also comprise cholesterol.Lipid-encapsulated RNA nanoparticles can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different RNAs that express one or more polypeptides.

[0212] In lipid-encapsulated RNA nanoparticles, RNA is completely encapsulated in the lipid portion of the particle, thereby protecting RNA from nuclease degradation.In a preferred embodiment, lipid-encapsulated RNA nanoparticles comprise RNA that is completely encapsulated in the lipid portion of the particle, thereby protecting RNA from nuclease degradation.In a specific example, the RNA in lipid particles is not substantially decomposed even after the particles are exposed to nuclease at 37 ℃ for at least 20, 30, 45 or 60 minutes.In another specific example, the RNA in lipid particles is not substantially decomposed even after the particles are incubated in serum at 37 ℃ for at least 30, 45 or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours.In another embodiment, RNA is complexed with the cationic lipid of lipid-encapsulated RNA nanoparticles. One advantage of the formulations of the present disclosure is that the lipid-encapsulated RNA nanoparticles are substantially non-toxic to mammals, such as humans.

[0213] In other embodiments, the present disclosure provides nucleic acid-lipid particle compositions comprising a plurality of nucleic acid-lipid particles.

[0214] Lipid particles contain RNA completely encapsulated within the lipid portion of the particle, resulting in 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 30%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95%, 80%-95%, 85%-95%, 90%-95%, 30%-90%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 30%-95%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 85%-100%, 90%-100%, 30%-10 ... 0%-90%, 50%-90%, 60%-90%, 70%-90%, 80%-90%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (or any fraction thereof or within that range) have RNA encapsulated therein.

[0215] Depending on the intended use of the lipid-encapsulated RNA nanoparticles, the ratio of components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0216] Dilution of lipid-encapsulated RNA nanoparticles After mixing organic lipid solution with aqueous RNA solution, further dilution of lipid-encapsulated RNA nanoparticle suspension before removing free RNA can increase the degree of RNA encapsulation.In one embodiment, the buffer solution can be 2 volumes of 15 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5, to reduce ethanol concentration to 8.25%.In another embodiment, the buffer solution can be 3 volumes of 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5, to reduce ethanol concentration to 6.25%.In another embodiment, the buffer solution can be 1 volume of 50 mM phosphate buffer, pH 6.0, to reduce ethanol concentration to 12.5%.In another embodiment, the buffer solution can be 2 volumes of 50 mM phosphate buffer, pH 6.0, to reduce ethanol concentration to 8.3%.In another embodiment, the buffer solution can be 3 volumes of 50 mM phosphate buffer, pH 6.0, to reduce ethanol concentration to 6.25%. In another embodiment, the buffer can be 3 volumes of 20 mM HEPES, 50 mM NaCl, 9% sucrose, pH 7.4 to reduce the ethanol concentration to 6.25%. In another embodiment, the buffer can be 1-3 volumes of 50 mM phosphate buffer, pH 6.0, 3 volumes of 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5 to reduce the ethanol concentration to 6.25%.

[0217] The diluted lipid-encapsulated RNA nanoparticles are then optionally collected in a container maintained at 15-20°C and incubated for a few minutes to 2 hours before a second dilution or concentration step in 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5.

[0218] Sample concentration The diluted lipid-encapsulated RNA nanoparticles can be concentrated, for example, by tangential flow filtration (TFF) using hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories, Inc., Rancho Dominguez, California), optionally via a peristaltic pump, a four-piston diaphragm pump, or a centrifugal pump (based on the principle of magnetic levitation). Methods for such concentration techniques are known in the art and readily apparent to those skilled in the art.

[0219] Removal of free RNA and replacement of buffer After concentration, the product may be diafiltered against 7-10 volumes of 110 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5 to remove organic solvent and unbound RNA. Preferably, the diafiltration buffer is added via a heat exchanger to maintain the product temperature at 15-20°C. The formulation may be further concentrated to achieve a total formulated RNA concentration of greater than 3 mg / mL.

[0220] Sterile filtration The RNA concentration in the lipid-encapsulated RNA nanoparticle formulation is then measured by IPRP-HPLC (ion-pair reverse-phase high-performance liquid chromatography). The final glycerol concentration in the formulation is adjusted to approximately 2 mg / mL (1.85-2.3 mg / mL) by diluting with 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5 containing glycerol so that the final glycerol concentration in the formulation is 5%. The diafiltered lipid-encapsulated RNA nanoparticles are filter-sterilized through a 0.2 µm sterilizing-grade filter (PES) at a lipid concentration of 56-69 mg / mL.

[0221] aseptic filling The filtered formulation is then aseptically filled into glass vials, stoppered, capped and placed at -20 or -70±5°C.

[0222] Device The present description provides an apparatus for carrying out the above manufacturing method. Figure 4 is a representative schematic diagram of an example of an apparatus according to one embodiment of the present description.

[0223] An aqueous solution containing RNA is transported by an HPLC pump through tubing, e.g., 0.03" ID PEEK tubing, 0.05" ID PEEK connectors, 0.0625" silicone tubing, 0.122" ID silicone tubing, and a 0.132" ID stainless steel section. An organic solution containing lipids is transported by an HPLC pump through tubing, e.g., 0.03" ID PEEK tubing, 0.02" ID PEEK connectors, and a 0.01" ID stainless steel section. The organic solution is pumped into the aqueous solution at a 90° angle in the mixing module. Thus, the organic solution containing lipids is introduced into the aqueous solution in a flow perpendicular to the flow of the aqueous solution. This introduction at a right angle to the flow direction occurs in a mixing module as shown in Figure 5, resulting in turbulent mixing under carefully controlled conditions to ensure that lipid nanoparticle encapsulation of RNA is formed in an acceptable manner in terms of particle size, dispersion, and encapsulation efficiency. The tube containing the mixed lipid-RNA is then transported to a second mixing zone, e.g., by a 0.25-inch ID polypropylene tube that meets the dilution buffer at a 45° angle with the dilution zone, and the diluted lipid-encapsulated RNA nanoparticles are collected in a stainless steel jacketed vessel maintained at 15-20°C. The particles are further processed, e.g., by tangential flow filtration using a diaphragm or centrifugal pump.

[0224] In one embodiment, the mixing region is a mixing module in which the organic lipid solution is delivered to the stream of aqueous RNA solution, preferably at an angle of about 90°. A first 0.132-inch (3.35 mm) ID stainless steel tube transporting the aqueous RNA solution has holes in the wall midway between both ends. A second 0.01-inch ID, 0.0625-inch OD tube is attached vertically by fitting into a hole in the wall of the first tube, allowing liquid to be transported from the second tube to the inside of the first tube (see Figure 5). In a preferred embodiment, lipid-encapsulated RNA nanoparticles of well-defined shape and reproducible size are prepared using a flow rate of the aqueous RNA solution that is preferably three times that of the organic lipid solution. Vesicles with well-defined shape and reproducible size can also be prepared, for example, by changing the flow rate of the fluid lines, in some cases to ensure sufficient mixing.

[0225] 5 illustrates a mixing module and associated fluid dynamics according to one embodiment. Compared to previous systems, the present disclosure provides turbulent flow and increased shear rates. For example, the present disclosure provides turbulent flow (N ) with shear rates of about 500 / sec to about 3300 / sec at flow rates (both flow lines) of about 0.1 L / min to about 0.3 L / min in the mixing environment. re >2000) advantageously.

[0226] The description herein provides a device with tangential flow filtration using hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories, Inc., Rancho Dominguez, California) and a four-piston diaphragm pump or centrifugal pump.

[0227] Pharmaceutical Composition The lipid-encapsulated RNA nanoparticles described herein are useful as components of pharmaceutical compositions.These compositions typically contain, in addition to lipid-encapsulated RNA nanoparticles, a pharmaceutically acceptable carrier.A detailed description of pharmaceutically acceptable carriers can be found in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472 (incorporated herein in its entirety).

[0228] The pharmaceutical compositions described herein may contain lipid nanoparticles in plain water (e.g., water for injection) or a buffer solution such as phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer. Buffer salts are typically present in the range of 5-20 mM.

[0229] The pharmaceutical compositions described herein may have a pH of 5.0 to 9.5, for example, 6.0 to 8.0.

[0230] The pharmaceutical compositions described herein may include a sodium salt (e.g., sodium chloride) to achieve isotonicity. The sodium salt may be NaCl at a concentration of 10±2 mg / ml, e.g., about 9 mg / ml.

[0231] The pharmaceutical compositions described herein may contain metal ion chelators. These can extend RNA stability by removing one or more ions that can accelerate phosphodiester hydrolysis, such as EDTA, EGTA, BAPTA, or pentetic acid. Such chelators are preferably present at a concentration of 10-500 μM, e.g., 0.1 mM. Citrate salts, such as sodium citrate, can also act as chelators, advantageously providing buffering activity.

[0232] The pharmaceutical compositions described herein preferably have an osmolality of 200 mOsm / kg to 400 mOsm / kg, for example 240 to 360 mOsm / kg or 290 to 310 mOsm / kg.

[0233] The pharmaceutical compositions described herein preferably contain one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are most preferred.

[0234] The pharmaceutical compositions described herein are preferably sterile.

[0235] The pharmaceutical compositions described herein are preferably non-pyrogenic, eg, having less than 1 EU (endotoxin unit, a standard measure) per dose, preferably less than 0.1 EU per dose.

[0236] The pharmaceutical compositions described herein are preferably gluten-free.

[0237] The pharmaceutical compositions described herein may be prepared in unit dose form. In some embodiments, the unit dose may have a volume of 0.1 to 1.0 mL, for example, about 0.5 mL.

[0238] The compositions may be prepared as injections, either as solutions or suspensions. The compositions may be prepared for pulmonary administration, for example, by inhaler, using a fine spray. The compositions may be prepared for administration to the nasal cavity, ear, or eye, for example, as a spray or drops.

[0239] The pharmaceutical composition comprises an immunologically effective amount of lipid nanoparticles, and optionally other optional ingredients.

[0240] The pharmaceutical compositions described herein are also suitable for administration by a delivery device, such as a syringe, nebulizer, sprayer, inhaler, or skin patch, which can be used to administer the composition to a vertebrate subject.

[0241] The lipid-encapsulated RNA nanoparticles described herein do not contain ribosomes.

[0242] definition The term "approximately" or "about" when applied to one or more values of interest refers to a value that is similar to the stated reference value.In certain embodiments, the term "approximately" or "about" refers to a range of values that falls within ±10% of the stated value, unless otherwise specified or clear from the context (except when such number exceeds 100% of possible values).

[0243] The terms "associated with," "conjugated," "linked," "attached," and "tethered," when used in reference to two or more moieties, mean that the moieties are physically associated or connected to one another, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. The "association" need not be strictly by direct covalent chemical bonding. It may also imply that ionic or hydrogen bonding, or hybridization-based connections, are sufficiently stable so that the "associated" entities remain physically associated.

[0244] In the claims, articles such as "a," "an," and "the" can mean one or more unless specifically stated to the contrary or clear from the context. A claim or specification containing "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless specifically stated to the contrary or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all members of a group are present in, employed in, or otherwise relevant to a given product or process.

[0245] The term "acyl," as used herein, refers to a hydrogen or alkyl group (e.g., a haloalkyl group), as defined herein, attached to the parent molecular group through a carbonyl group, as defined herein, and includes, for example, formyl (i.e., a carboxaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl, and the like. Exemplary unsubstituted acyl groups contain 1 to 7, 1 to 11, or 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents, as described herein.

[0246] The term "alkenyl," as used herein, unless otherwise specified, refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms (e.g., 2 to 6 or 2 to 10 carbon atoms) containing one or more carbon-carbon double bonds, and examples include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkyl includes both cis and trans isomers. Alkyl groups can be optionally substituted with one, two, three, or four substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0247] The term "alkoxy", unless otherwise specified, refers to a chemical substituent of formula -OR, where R is C 1-20 Alkyl groups (e.g., C 1-6 or C 1-10 Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents (e.g., hydroxy or alkoxy) as defined herein.

[0248] As used herein, unless otherwise specified, the term "alkyl" includes both straight-chain and branched-chain saturated groups having 1 to 20 (e.g., 1 to 10 or 1 to 6) carbon atoms. Examples of alkyl groups include methyl, ethyl, n- and isopropyl, n-, sec-, iso-, and tert-butyl, neopentyl, and the like. The term "lower alkyl" refers to a group having 1 to 6 carbons in the chain, which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0249] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, and the like. Alkyl groups can be optionally substituted with one, two, three, or four substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0250] The term "amphipathic lipid" or "amphiphilic lipid" refers to a lipid material in which the hydrophobic portion orients toward the hydrophobic phase and the hydrophilic portion orients toward the aqueous phase. The hydrophilic properties result from the presence of polar or charged groups, such as carbohydrates, phosphates, carboxylic acids, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0251] The term "anionic lipid" refers to a lipid that is negatively charged at physiological pH, including, but not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and palmitoyloleoylphosphatidylglycerol (POPG).

[0252] "Antisense" is a polynucleotide that interferes with the function of DNA and / or RNA. This can result in the inhibition of expression.

[0253] "Aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0254] The term "back pressure" refers to the resistance or force that opposes the desired flow of a fluid through a conduit, resulting in frictional losses and pressure drop. Fluid tends to be directed and flow away from areas of high pressure and toward areas of low pressure.

[0255] The term "comprising" is intended to be open-ended, permitting but not requiring the inclusion of additional elements or steps. Thus, where the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0256] The term "composition" means something comprising the specified ingredients in the specified amounts, as well as anything that results directly or indirectly from combining the specified ingredients in the specified amounts.

[0257] The term "commercially available chemicals" and the chemicals used in the examples described herein are available from standard commercial sources, such as Acros Organics (Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0258] The term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0259] " Completely encapsulated " means that the RNA in nucleic acid-lipid particles is not significantly degraded after being exposed to serum or nuclease assay, which significantly degrades free RNA.When completely encapsulated, in the process that usually degrades 100% of free nucleic acid, less than 25% of the nucleic acid in particles is degraded, more preferably less than 10%, most preferably less than 5%." Completely encapsulated " also means that when administered in vivo, nucleic acid-lipid particles are not rapidly degraded into their components.

[0260] In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay using a dye whose fluorescence increases when bound to nucleic acids. Encapsulation is determined by adding the dye to the liposome formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed when a small amount of nonionic surfactant is added. Disruption of the liposome bilayer via surfactant releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation can be calculated as E = (I0 - I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of surfactant.

[0261] "Gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e.g., herpes simplex virus). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional property (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length polypeptide or a fragment thereof is retained.

[0262] The term "hydrophobic lipid" refers to a compound that has a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and optionally substituted with one or more aromatic groups, alicyclic groups or heterocyclic groups.Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

[0263] An "immunologically effective" amount or dose is one that is sufficient to elicit an immunological response in a subject, thereby building immunity in the subject. Immunity can be confirmed, for example, by antibody titer testing to determine whether sufficient antibodies against a particular infectious disease are detected in the subject.

[0264] "Lamellar morphology" refers to a bilayer structure. The lamellar morphology, bilayer structure of the lipid particles described herein can be determined using analytical techniques, for example, by cryo-TEM imaging.

[0265] The term "lipid" refers to organic compounds that contain esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0266] The term " lipid conjugate " refers to the conjugated lipid that prevents particle aggregation.Such lipid conjugate includes but is not limited to PEG-lipid conjugate, for example, PEG that is bound to dialkyloxypropyl (for example, PEG-DAA conjugate), PEG that is bound to diacylglycerol (for example, PEG-DAG conjugate), PEG that is bound to cholesterol, PEG that is bound to phosphatidylethanolamine and PEG that is bound to ceramide, cationic PEG lipid, polyoxazoline (POZ)-lipid conjugate, polyamide oligomer, and their mixture.PEG or POZ can be directly bound to lipid, or can be bound to lipid via linker moiety.Any suitable linker moiety for binding PEG or POZ to lipid can be used, for example, includes non-ester-containing linker moiety and ester-containing linker moiety.In certain preferred embodiments, non-ester-containing linker moiety such as amide or carbamate is used.

[0267] The term "lipid delivery vehicle" refers to a lipid formulation that can be used to deliver therapeutic nucleic acid (e.g., mRNA) to a target site (e.g., cell, tissue, organ, etc.). The lipid delivery vehicle can be a nucleic acid-lipid particle that can be formed from cationic lipid, non-cationic lipid (e.g., phospholipid), conjugated lipid that prevents particle aggregation (e.g., PEG-lipid), and optionally cholesterol. Typically, therapeutic nucleic acid (e.g., mRNA) is encapsulated in the lipid portion of the particle, which can protect the particle from enzymatic degradation.

[0268] "Lipid nanoparticles" are any lipid compositions that can be used to deliver compounds, including, but not limited to, liposomes that contain a lipid bilayer, either as a single layer or a multilayer structure, in which RNA is at least partially encapsulated by ion pairing with a cationic lipid. When the lipid nanoparticle is a liposome, it is considered to have an aqueous interior. Other lipid nanoparticles have a solid interior in which a lipid layer (which can be a bilayer or a monolayer) is directly bound to the encapsulated substance (eg, nucleic acid).

[0269] "Lipid encapsulation" can refer to a lipid formulation that provides a fully encapsulated, partially encapsulated, or both, compound in which the RNA (or another nucleic acid) is inaccessible to RNase-mediated hydrolysis or dye intercalation.

[0270] The term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein or polypeptide of interest and can be translated to produce the encoded protein or polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0271] The term "neutral lipid" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0272] The term "non-cationic lipid" refers to an amphipathic lipid, a neutral lipid, or an anionic lipid as described herein.

[0273] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and unnatural, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0274] The term "nucleotide" is intended to include nucleotides having natural (standard) or modified bases known in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moieties (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and the like; see, e.g., Usman and McSwiggen, supra; Eckstein et al., WO 92 / 07065; Usman et al., WO 93 / 15187; Uhlman and Peyman, supra, all incorporated herein by reference). Some examples of modified nucleobases known in the art are summarized in Limbach, et al., Nucleic Acids Res. 22:2183, 1994. Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). "Modified base" in this embodiment refers to a nucleotide base other than adenine, guanine, cytosine, and uracil at the 1' position, or their equivalents.

[0275] "Organic lipid solution" refers to a composition comprising, in whole or in part, an organic solvent with lipids. The organic lipid solution preferably comprises an alkanol, most preferably ethanol.

[0276] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0277] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) other than the compounds described herein that has the properties of being substantially non-toxic and non-inflammatory in patients. Excipients can include, for example, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, excipients (diluents), film-forming or coating agents, flavors, fragrances, flow agents (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary additives include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), citric acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0278] The phrase "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound has been modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfonate. Salts include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG 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.

[0279] "Ribonucleic acid" or "RNA" refers to a polymer containing at least two ribonucleotides. A "ribonucleotide" contains the sugar ribose, a base, and a phosphate group. The nucleotides are linked via the phosphate group. "Bases" include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and purines and pyrimidines, including further naturally occurring analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylic acids, and alkyl halides.

[0280] The RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), self-replicating RNA, ribozyme, chimeric sequence, or derivatives of these groups. The RNA may contain one or more nucleotides with modified nucleobases (in addition to any 5' cap structure), such as m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-methyladenosine); Isopentenyladenosine; ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A( N6-Methyl-N6-threonylcarbamoyladenosine; hn6A (N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine); m'Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-phenylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl2O-methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine);m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (quoiosine); oQ (epoxyquoiosine); galQ (galactosyl-quoiosine); manQ (mannosyl-quoiosine); preQo (7-cyano-7-deazaguanosine) );preQi(7-aminomethyl-7-deazaguanosine);G*(archaeosine);D(dihydrouridine);m5Um(5,2'-O-dimethyluridine);s4U(4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);ho5U(5-hydroxyuridine);mo5U(5-methoxyuridine);cmo5U(uridine 5-oxyacetic acid);mcmo5U(uridine 5-oxyacetic acid methyl ester) terephthalate);chm5U (5-(carboxyhydroxymethyl)uridine));mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester);mcm5U (5-methoxycarbonylmethyluridine);mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine);nm5s2U (5-aminomethyl-2-thiouridine);mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine);m nm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine);m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,TO-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl m1Gm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine) irinomethyluridine; tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil uracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine , N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or basic nucleotide;

[0281] The RNA may comprise one or more UNA molecules, for example, as disclosed in U.S. Patent Nos. 8,314,227, 9,051,570, 9,303,260, 9,297,009 and 9,340,789, and U.S. Patent Application Publication No. 2016 / 0168567 (incorporated herein in its entirety).

[0282] The RNA or self-replicating RNA may contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues.

[0283] "Self-replicating RNA" refers to RNA that, when released from a mammalian cell in the absence of protein, can result in the production of multiple daughter RNAs by its own transcription (by generating antisense copies of itself). Self-replicating RNA molecules are typically positive-strand molecules that can be directly translated upon release into the cell; this translation provides an RNA-dependent RNA polymerase that generates both antisense and sense transcripts of the released RNA. The daughter RNAs, and collinear subgenomic transcripts, can be translated themselves to provide expression of the encoded protein (e.g., antigen or immunogen) in situ, or can be transcribed to provide additional transcripts synonymous with the RNA that is translated to provide protein expression in situ. The overall result of this transcriptional sequence is a large amplification of the number of introduced RNA replicons, and thus the encoded protein becomes the primary product in transfected cells. Examples of self-replicating RNAs are described in WO2012 / 006369 and US2018 / 0104359, the contents of which are incorporated by reference.

[0284] "Turbulent flow" is defined herein in terms of the shape of the mixing region and the flow rate of the aqueous solution containing RNA and the ethanol solution containing the lipid mixture. During mixing, the Reynolds number is at least 2000, and the Reynolds number Re is defined as follows: Re=DVρ / μ where ρ is the density (g / cm) of a solution containing 0–25% ethanol in water.3 ), V is the velocity of the solution relative to the tube (cm / s), D is the ID of the tube (cm), and μ is the dynamic viscosity of the solution (g / (cm·s)). Tilton, Fluid and Particle Dynamics, PERRY'S CHEMICAL ENGINEERS' HANDBOOK (Green ed., 8 th ed. 2008) (incorporated in its entirety), pp. 6-51. The transition to turbulence occurs at Reynolds numbers Re in the range of 2000-2500 (Tilton, 6-14). For example, at a flow rate of 300 ml / min in a tube with ID = 0.132 in, Re is 2100 (D = 0.30 cm, V = 70 cm / sec, ρ = 96 g / cm 3 and μ = 0.01 g / (cm·sec) (Poling, Physical and Chemical Data, in PERRY'S, pp. 2-117 and 2-448). Considering the velocity profile of the tube, the centerline velocity v as a function of radial position r in a circular tube of radius R(D / 2) is defined as: v=2V(1-r 2 / R 2 ) where the maximum velocity is twice the effective velocity for a parabolic profile (Tilton, 6-11). For example, at a flow rate of 300 ml / min, the Reynolds number at the center of the tube is 4,200. Therefore, it is possible to determine the flow velocity at which turbulence toward the wall occurs to a significant degree, at least in the center of the tube. [Example]

[0285] Example 1: Effect of flow rate on particle size As shown in Figure 1, a reservoir containing siRNA at a concentration of 0.55 mg / mL and another reservoir containing total lipid at a concentration of 49.4 mg / mL were prepared.

[0286] Lipid compositions were formulated in the range of 48-60:5-10:28-38:0.5-3.0 mol% cationic lipid:DSPC:cholesterol:PEG-DMG. The total lipid to RNA ratio was 25:1-30:1 (wt:wt).

[0287] The lipid solution was pumped from a reservoir through the mixing module by an HPLC pump through tubing with a 0.01-inch lipid inlet at a flow rate of 25 to 87.5 ml / min. The siRNA solution was pumped from a reservoir through the module by an HPLC pump through tubing with a 0.132-inch siRNA inlet. The siRNA flow rate was three times that of the lipid flow rate, and the mixture outlet was a 25% EtOH solution. The outlet solution was further diluted with buffer, and free RNA and ethanol were removed by tangential flow filtration (TFF). Figures 1, 4, and 5 show schematics of the overall manufacturing method, with Figure 1 outlining the manufacturing steps, Figure 4 showing a schematic of the apparatus, and Figure 5 providing a diagram of the mixing module of the present disclosure.

[0288] Various properties of the lipid-encapsulated RNA nanoparticles were measured, including the mean particle size (nm), polydispersity index (PDI), and encapsulation rate. For all conditions tested, the formulation yield was greater than 80%. The results are shown in Table 2. [Table 2]

[0289] Results showed that increasing the lipid and RNA flow rates decreased the mean particle size while maintaining a PDI of <0.1 and encapsulation at approximately 99%.

[0290] Example 2: Scalability and reproducibility of the manufacturing method Following the conditions of Example 1, a combined flow rate of 300 ml / min was used for batch volumes of 1 L to 220 L. The results are shown in Table 3. [Table 3]

[0291] The results demonstrate that the manufacturing process described herein using turbulent flow is scalable from 1.8 L to 220 L. Important physicochemical and biological properties remain unchanged upon scaling. Figure 7 provides cryo-TEM (transmission electron microscopy) micrographs showing that the particles produced are uniform in size and have a monolayer morphology in the 220 L batch.

[0292] The scalability of the manufacturing method described herein was measured by varying the amount of RNA processed from 0.05 to 30 g at a flow rate of 300 ml / min using the conditions described in Example 1 and measuring particle size, PDI, and encapsulation efficiency. The results are shown in Figure 6A. The results show that the particle size remained between 70 and 80 nm during scale-up from 0.5 g to 30 g.

[0293] The reproducibility of the manufacturing process was measured by preparing several batches of 30 g of RNA-lipid nanoparticles. The results are shown in Figure 6B. The results demonstrate batch-to-batch reproducibility, resulting in particle sizes of 70-80 nm.

[0294] The final formulation yield was over 80% at all scales from 1.8 to 220 L.

[0295] Efficacy of particles produced in a 220 L batch by the manufacturing method described herein via iv injection into the tail vein of Balb / c mice. Results are shown in Table 4. [Table 4]

[0296] The results show that lipid-encapsulated RNA nanoparticles produced by turbulent flow are well tolerated at doses up to at least 20 mg / kg.

[0297] Example 3: Effect of varying siRNA concentration The method of Example 1 was used with lipid flow rate of 75ml / min, siRNA flow rate of 225ml / min and total flow rate of 300ml / min.The concentration of siRNA in the manufacturing process before dilution was changed from 0.083mg / ml to 0.41mg / ml, and the concentration of lipid was increased proportionally to maintain the RNA:lipid ratio.The results are shown in Table 5. [Table 5]

[0298] The results showed that the concentration of RNA during the manufacturing process could be varied without changing the particle size or encapsulation rate of the RNA.

[0299] Example 4: Effect of module size A 0.03 RNA:lipid (wt:wt) ratio was used, following the conditions of Example 1. The effect of varying the module inlet diameter was measured by varying the overall flow rate from 40 to 600 ml / min, as shown in the results in Table 6. The inlet pressure was monitored, and the size (PDI) and encapsulation efficiency of the resulting lipid-encapsulated RNA particles were measured. [Table 6]

[0300] The results show that nanoparticles of various sizes can be produced with an encapsulation efficiency of over 99% and a yield of over 80% by simply changing the ID of the lipid and / or siRNA inlet in the mixing module without changing the composition.

[0301] Example 5: Preparation of lipid-encapsulated RNA nanoparticles containing mRNA or self-replicating RNA Following the conditions of Example 1, a module with a 0.01-inch lipid inlet and a 0.132-inch siRNA inlet, a lipid flow rate of 75 ml / min, an mRNA flow rate of 225 ml / min, and an overall flow rate of 300 ml / min was used. Single-stranded mRNAs of various sizes, ranging from 265 kDa to 3,858 kDa, were used, while maintaining the RNA / total lipid (wt / wt) ratio between 0.025 and 0.035. The RNAs used were mRNAs containing replicon regions or self-replicating RNAs. [Table 7]

[0302] The results shown in Table 7 demonstrate that mRNAs of approximately 0.8-12 kilobases (kb) can be packaged with approximately 95-97% encapsulation using the manufacturing methods described herein. The size of the resulting particles only increases with mRNA lengths greater than 5.5 kb.

[0303] Example 6: Generation of lipid-encapsulated RNA nanoparticles containing mRNA or self-replicating RNA Following the conditions of Example 1, a module with a 0.01-inch lipid inlet and a 0.132-inch siRNA inlet, a lipid flow rate of 75 ml / min, an mRNA flow rate of 225 ml / min, and an overall flow rate of 300 ml / min was used. Single-stranded mRNAs of various sizes, ranging from 265 kDa to 3,858 kDa, were used, while maintaining the RNA / total lipid (wt / wt) ratio between 0.025 and 0.035. The RNAs used were mRNAs containing replicon regions or self-replicating RNAs.

[0304] Example 6: Bioactivity of lipid-encapsulated nanoparticles in vivo Lipid-encapsulated RNA nanoparticles containing either EPO mRNA or FVII siRNA were formulated using the manufacturing method described herein and then injected into Balb / c mice (6-8 weeks old). The bioactivity of these nanoparticles was then measured by assessing the levels of erythropoietin (EPO) and FVII protein in the serum or plasma of the mice.

[0305] Using liver-directed in vivo screening of a lipid library, we tested a series of compounds that promote high levels of siRNA-mediated gene silencing in hepatocytes, the cells that make up the liver parenchyma. Factor VII, a blood coagulation factor, is a suitable target gene for assaying functional siRNA delivery to the liver. Because this factor is specifically produced in hepatocytes, gene silencing indicates successful delivery to the parenchyma, as opposed to delivery to cells of the reticuloendothelial system (e.g., Kupffer cells). Furthermore, factor VII is a secreted protein and can be easily measured in serum, eliminating the need for animal euthanasia. Silencing at the mRNA level can be easily determined by measuring protein levels. This is due to the protein's short half-life (2-5 hours). A composition containing siRNA directed against factor VII was formulated. Female C57BL / 6 mice (6-8 weeks old) were used for FVII siRNA knockdown (KD) studies.

[0306] The manufacturing method of Example 1 was followed. In vivo biological activity in mice was measured as described above. The results are shown in Table 8. [Table 8]

[0307] The results showed that both EPO mRNA-mediated expression and FVII siRNA-mediated knockdown (KD) of FVII expression were potent in lipid-encapsulated RNA nanoparticles containing either EPO mRNA or FVII siRNA formulated using the manufacturing methods described herein.

[0308] Example 7: In vivo bioactivity of lipid-encapsulated mRNA particles with different lipid compositions Lipid-encapsulated RNA nanoparticles containing EPO mRNA with varying lipid compositions were formulated using the manufacturing method described herein. The ionizable cationic lipid was varied: Lipid 1 in Table 1 was compared with Lipid 2, Lipid 3, and Lipid 9. The structures of the ionizable cationic lipids are shown below. The formulation compositions are shown in Table 9. [ka]

[0309] Serum EPO protein expression (ng / ml) was measured after a single administration of 0.3 mg / kg mRNA to female Balb / c mice (6-8 weeks old). The PBS negative control showed EPO expression of 2 ng / ml. Comparative results are shown in Table 9. [Table 9]

[0310] The results showed that although particle sizes were comparable, EPO protein expression varied significantly between lipid-encapsulated EPO mRNA nanoparticles with different ionizable cationic lipids and compositions. All lipid-encapsulated EPO mRNA nanoparticles produced using the manufacturing methods described herein were potent. Yields of greater than 85% were observed for the compositions tested.

[0311] Further considerations The foregoing description is provided to enable one skilled in the art to implement the various configurations described herein. There may be many other ways of implementing the subject technology. The various functions and elements described herein may be divided differently than shown without departing from the scope of the subject technology. Various modifications to these configurations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other configurations. Thus, many changes and modifications may be made to the subject technology by those skilled in the art without departing from the scope of the subject technology.

[0312] While the detailed description contains many specific details, these should not be construed as limiting the scope of the subject technology, but merely as illustrating various examples and embodiments of the subject technology. It should be understood that the scope of the subject technology includes other embodiments not described in detail above. Various other modifications, changes, and variations may be made in the arrangement, operation, and details of the methods and apparatus of the subject technology disclosed herein without departing from the scope of the present disclosure. Moreover, it is not necessary for a device or method to address every problem that can be solved (or have every advantage that can be achieved) by different embodiments of the present disclosure to be within the scope of the present disclosure. The use of "can" and its derivatives herein should be understood in the sense of "may" or "optionally," as opposed to a positive ability.

Claims

1. 1. An apparatus for producing lipid-encapsulated RNA nanoparticles, the apparatus comprising: (a) a first HPLC pump connected to the mixing module, the first HPLC pump configured to pump an aqueous solution comprising RNA at a flow rate of at least 150 mL / min; (b) a first reservoir connected to a first HPLC pump, the first reservoir containing an aqueous solution; (c) a second HPLC pump connected to the mixing module, the second HPLC pump configured to pump an ethanol solution containing one or more lipids at a flow rate greater than 50 mL / min; (d) a second reservoir connected to a second HPLC pump, the second reservoir containing an ethanol solution; Including, the mixing module includes a first tube configured to receive an aqueous solution and a second tube configured to receive an ethanol solution, the first tube having an inner diameter (ID) of 0.1 inches to 0.132 inches and the second tube having an inner diameter (ID) of 0.005 inches to 0.02 inches; the second tube extends perpendicularly through the wall of the first tube and partially into the interior of the first mixing tube; the apparatus is configured to mix the ethanol solution with the aqueous solution by introducing the ethanol solution into the aqueous solution at a region within the mixing module to generate an exit solution having a flow that generates turbulence; The one or more lipids have a pKa of about 6 to about 7 and a structure represented by Formula I: 【Chemistry 1】 (I) [In the formula, R 5 and R 6 each independently being a straight-chain or branched-chain C 1- C 31 Alkyl, C 2- C 31 Alkenyl or C 2- C 31 selected from the group consisting of alkynyl and cholesteryl; L 5 and L 6 are each independently a linear C 1- C 20 Alkyl and C 2- C 20 alkenyl; X 5 is —C(O)O— or —OC(O)—; X 6 is —C(O)O— or —OC(O)—; X 7 is S or O; L 7 is absent or is lower alkyl; R 4 but linear or branched C 1- C 6 is alkyl; and R 7 and R 8 each independently represents hydrogen and a straight or branched chain C 1- C 6 alkyl] A device comprising a cationic lipid having the structure shown in Figure 1 or a pharmaceutically acceptable salt or solvate thereof.

2. 10. The apparatus of claim 1, wherein the first tube and the second tube are stainless steel tubes.

3. 3. The apparatus of claim 1, wherein a first HPLC pump is connected to a first tube of the mixing module through one tube and a second HPLC pump is connected to a second tube of the mixing module through another tube.

4. 4. The apparatus of claim 3, wherein the tubing connecting the first HPLC pump to the first tubing of the mixing module and the other tubing connecting the second HPLC pump to the second tubing of the mixing module are each made of polyetheretherketone (PEEK).

5. 5. The apparatus of claim 3 or 4, wherein the other tubing connecting the second HPLC pump to the mixing module has an ID of 0.02 inches to 0.08 inches or 0.03 inches to 0.04 inches, and the tubing connecting the first HPLC pump to the mixing module has an ID of 0.02 inches to 0.04 inches or 0.03 inches.

6. 6. The apparatus of any one of claims 1 to 5, wherein the first HPLC pump is configured to pump the aqueous solution comprising RNA at a flow rate of at least 225 mL / min, at least 300 mL / min, at least 450 mL / min, or between about 200 mL / min and about 450 mL / min, and the second HPLC pump is configured to pump the ethanol solution at a flow rate of more than 75 mL / min, more than 100 mL / min, or more than 150 mL / min, or between about 50 mL / min and about 150 mL / min.

7. 7. The apparatus of any one of claims 1 to 6, wherein the ethanol solution is pumped at a flow rate that is one-third of the flow rate of the aqueous solution.

8. 8. The device of any one of claims 1 to 7, wherein the draining solution has a flow rate of at least 200 mL / min, at least 300 mL / min, or at least 600 mL / min.

9. 9. The apparatus of claim 1, wherein the apparatus is configured to produce an output solution flowing through the first tube comprising a turbulent flow of RNA and one or more lipids in about 10% to 75% v / v ethanol.

10. The device of any one of claims 1 to 9, wherein the lipid-encapsulated RNA nanoparticles have a bilayer structure.

11. 11. The device of any one of claims 1 to 10, wherein the RNA is greater than 98% encapsulated.

12. 12. The device of any one of claims 1 to 11, wherein the lipid-encapsulated RNA nanoparticles have a polydispersity index of 0.09 or less.

13. 13. The apparatus of any one of claims 1 to 12, wherein the first HPLC pump is configured to pump the aqueous solution through the first tube with a back pressure of at least 10 psi, 25 psi, 50 psi, 75 psi or 100 psi, and the second HPLC pump is configured to pump the ethanol solution through the second tube with a back pressure of at least 40 psi, 80 psi, 150 psi, 300 psi or 400 psi.

14. 14. The device of any one of claims 1-13, wherein the first tube has an ID of 0.132 inches and the second tube has an ID of 0.007 inches, 0.01 inches, or 0.02 inches.

15. 15. The apparatus of any one of claims 1 to 14, wherein the apparatus is configured to maintain the aqueous solution, the ethanol solution and the effluent solution at a temperature of about 15 to 20°C.

16. 16. The apparatus of any one of claims 1 to 15, further comprising a third tube connected to a third HPLC pump configured to pump the dilution buffer by introducing the dilution buffer into the outlet solution in the region of a Y-connector connecting the third tube to the first tube, thereby mixing the dilution buffer with the outlet solution to produce a diluted outlet solution.

17. 17. The apparatus of claim 16, wherein the third HPLC pump is configured to pump the dilution buffer through the third tube at a flow rate of 400 to 900 mL / min.

18. 18. The apparatus of claim 16 or 17, wherein the third tube has an ID of 0.25 inches.

19. 19. The device of any one of claims 1 to 18, wherein the lipid-encapsulated RNA nanoparticles have an average particle size of less than about 100 nm.

20. 20. The device of any one of claims 1 to 19, wherein the lipid portion of the lipid-encapsulated RNA nanoparticles comprises about 48 mol% to about 66 mol% cationic lipid, about 2 mol% to about 12 mol% DSPC, about 25 mol% to about 42 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG.

21. 21. The device of any one of claims 1 to 20, wherein the lipid-encapsulated RNA nanoparticles have a total lipid:RNA weight ratio of about 50:1 to about 3:

1.

22. Lipid-encapsulated RNA nanoparticles were 25:25:10:38.5:1.5, 25:25:10:37:3, 25:25:10:35:5, 20:20:7:51.5:1.5, 25:20:10:42:3, 20:30:13:32:5, 25:20:10:40:5, 25:25:13:35.5:1.5, 25:30:7:35:3, 30:20:13:34:3, 30:25:7:33:3, 30:30:10:25.8:1.5, 15:20:13 22. The device of any one of claims 1 to 21, comprising a molar ratio of cationic lipid selected from among 20:49:3, 20:20:13:44:3, 20:25:13:39:3, 15:25:13:44:3, 20:25:13:39:3, 25:25:13:34:3, 30:20:13:34:3 and 30:30:13:29:3:DOTAP (1,2-dioleoyl-3-trimethylammonium-propane):DSPC:cholesterol:PEG.

23. 23. The device of any one of claims 1 to 22, wherein the lipid-encapsulated RNA nanoparticles comprise about 20 w / w% to 60 w / w% cationic lipid, about 5 w / w% to 30 w / w% helper lipid, about 0 w / w% to 60 w / w% cholesterol, and about 0.5 w / w% to 15 w / w% polyethylene glycol-lipid conjugate.

24. 24. The device of any one of claims 1 to 23, wherein the RNA is selected from the group consisting of transfer RNA, small nuclear RNA, ribosomal RNA, messenger RNA, antisense RNA, small interfering RNA and self-replicating RNA.

Images