Method of making lipid-encapsulated RNA nanoparticles

1. A method of producing a lipid-encapsulated RNA nanoparticle, comprising the steps

a) flowing an aqueous solution comprising an RNA through a 1 st tube having an inner diameter (ID) of between about 0.1″ and 0.132″;

b) flowing an ethanol solution comprising lipids through a 2 nd tube having an ID of between about 0.005″ and 0.02″ at one third the flow rate of the aqueous solution through the 1 st 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 2 nd tube perpendicularly joined to the 1 st tube;

wherein the mixing produces an output solution flowing in the 1 st tube comprising a turbulent flow of the RNA and the lipids in between about 10% to 75% ethanol v/v;

wherein the lipid-encapsulated RNA nanoparticles have a bilayer structure;

wherein the RNA is encapsulated at greater than 98%;

wherein the polydispersity index is less than 0.09; and,

wherein the cationic lipid has a pKa of about 6 to about 7 and a structure of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein

R 5 and R 6 are each independently selected from the group consisting of a linear or branched C 1 -C 31 alkyl, C 2 -C 31 alkenyl or C 2 -C 31 alkynyl and cholesteryl;

L 5 and L 6 are each independently selected from the group consisting of 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 C 1 -C 6 alkyl; and

R 7 and R 8 are each independently selected from the group consisting of a hydrogen and a linear or branched C 1 -C 6 alkyl.

2. The method of claim 1 , wherein the output has a flow rate of at least 200 ml/min.

3. The method of claim 1 , wherein the flow rate of the output has a Reynolds number of at least 2,000.

4. The method of claim 1 , wherein the aqueous solution is pumped through the 1 st tube by a 1 st HPLC pump with a back pressure of at least 10 psi, 25 psi, 50 psi, 75 psi, or 100 psi and the ethanol solution is pumped through the 2 nd tube by a 2 nd HPLC pump with a back pressure of at least 40 psi, 80 psi, 150 psi, 300 psi, or 400 psi.

5. The method of claim 1 , wherein the 1 st tube has an ID of 0.132″ and the 2 nd tube has an ID of 0.007″, 0.01″, or 0.02″.

6. The method of claim 1 , wherein 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; 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.

7. The method of claim 1 , wherein the aqueous, ethanol, output solutions are maintained at 15-20° C.

8. The method of claim 1 , wherein the mixing module is made of stainless steel and consists of the 2 nd tube mounted perpendicularly on the 1 st tube, wherein the 1 st tube has an opening through a wall, wherein the opening is the size of the outside diameter of the 2 nd tube, and wherein the 2 nd tube is fitted over the opening to permit continuous movement of the ethanol solution in the 2 nd tube into the aqueous solution in the 1 st tube.

9. The method of claim 1 , further comprising pumping a dilution buffer through a 3 rd tube, and mixing the dilution buffer with the output solution by introducing the dilution buffer to the output solution in the region of a Y-connector connecting the 3 rd tube to the 1 st tube to produce a diluted output solution.

10. The method of claim 9 , wherein the dilution buffer comprises

a) between about 10 mM and 20 mM Tris buffer, between about 45 mM and 55 mM NaCl, and between about 8% and 10% sucrose, at a pH between about 7.4 and 8.5; or

b) between 40 mM and 90 mM phosphate buffer, at a pH between about 6.0 and 6.5; and (i) the 3 rd dilution buffer comprises between about 20 mM and 50 mM HEPES buffer; or

(ii) the 3 rd dilution buffer comprises between 20 mM and 50 mM HEPES buffer, between about 50 mM and 300 mM NaCl, between about 0% and 15% sucrose, at a pH between about 7.4 and 8.5.

11. The method of claim 9 , wherein the diluted output solution comprises 6.25% ethanol; 8.25% ethanol; 8.3% ethanol; or 12.5% ethanol.

12. The method of claim 9 , wherein the dilution buffer is pumped through the 3 rd tube at a flow rate of 400-900 mL/min.

13. The method of claim 9 , wherein the 3 rd tube has an ID of 0.25″.

14. The method of claim 1 , wherein the lipid-encapsulated RNA nanoparticle has an average particle size of less than about 100 nm.

15. The method of claim 1 , wherein the lipid portion of the lipid-encapsulated RNA nanoparticle comprises about 48 mol % to about 66 mol % of a 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.

16. The method of claim 1 , wherein the lipid-encapsulated RNA nanoparticle has a total lipid:RNA weight ratio of about 50:1 to about 3:1.

17. The method of claim 1 , wherein the lipid-encapsulated RNA nanoparticle comprises a molar ratio of cationic lipid:DOTAP (1,2-dioleoyl-3-trimethylammonium-propane):DSPC:cholesterol:PEG selected from 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.

18. The method of claim 1 , wherein the lipid-encapsulated RNA nanoparticle comprises between about 20 w/w % and 60 w/w % of the cationic lipid, between about 5 w/w % and 30 w/w % of a helper lipid, between about 0 w/w % and 60 w/w % of a cholesterol, and between about 0.5 w/w % and 15 w/w % of a polyethylene glycol-lipid conjugate (PEG).

19. The method of claim 1 , wherein the RNA is selected from the group consisting of a transfer RNA, small nuclear RNA, ribosomal RNA, messenger RNA, antisense RNA, small interfering RNA and self-replicating RNA.