METHOD OF MAKING LIPID-ENCAPSULATED RNA NANOPARTICLES

1 - 20 . (canceled)

21 . An apparatus for producing a lipid-encapsulated RNA nanoparticle, comprising:

(a) an aqueous feed tube connected at one end to a 1 st HPLC pump and at the other end to a mixing module, wherein the 1 st HPLC pump is configured to pump an aqueous solution comprising RNA through the aqueous feed tube at a flow rate of at least 150 mL/min;

(b) a 1 st reservoir connected to the 1 st HPLC pump, wherein the 1 st reservoir contains the aqueous solution;

(c) an ethanol feed tube connected at one end to a 2 nd HPLC pump and at the other end to the mixing module, wherein the 2 nd HPLC pump is configured to pump an ethanol solution comprising one or more lipids through the ethanol feed tube at a flow rate greater than 50 mL/min,

(d) a 2 nd reservoir connected to the 2 nd HPLC pump, wherein the 2 nd reservoir contains the ethanol solution,

wherein the mixing module comprises a 1 st mixing tube configured to receive the aqueous solution from the aqueous feed tube, and a 2 nd mixing tube configured to receive the ethanol solution from the ethanol feed tube, the 1 st mixing tube having an inner diameter (ID) of 0.1″ to 0.132″, the 2 nd mixing tube having an ID of 0.005″ to 0.02″,

wherein the 2 nd mixing tube extends perpendicularly through a wall of the 1 st mixing tube and partly into the interior of the 1 st mixing tube,

wherein the apparatus is configured to mix the ethanol solution with the aqueous solution by introducing the ethanol solution into the aqueous solution in a region within the mixing module to produce an output solution having a flow that produces turbulence;

wherein the one or more lipids comprise a cationic lipid having 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.

22 . The apparatus of claim 21 , wherein the 1 st mixing tube and the 2 nd mixing tube are stainless steel mixing tubes.

23 . The apparatus of claim 21 , wherein the aqueous feed tube and the ethanol feed tube are polyether ether ketone (PEEK) tubes.

24 . The apparatus of claim 21 , wherein the aqueous feed tube has an ID of 0.02″ to 0.08″, and the ethanol feed tube has an ID of 0.02″ to 0.04″.

25 . The apparatus of claim 21 , wherein the aqueous feed tube has an ID of 0.03″ to 0.04″, and the ethanol feed tube has an ID of 0.03″.

26 . The apparatus of claim 21 , wherein the 1 st HPLC pump is configured to pump an aqueous solution comprising RNA through the aqueous feed tube at a flow rate of at least 225 mL/min, at least 300 mL/min, or at least 450 mL/min, and wherein the 2 nd HPLC pump is configured to pump the ethanol solution through the ethanol feed tube at a flow rate greater than 75 mL/min, 100 mL/min, or 150 mL/min.

27 . The apparatus of claim 21 , wherein the 1 st HPLC pump is configured to pump an aqueous solution comprising RNA through the aqueous feed tube at a flow rate of about 150 mL/min to about 450 mL/min.

28 . The apparatus of claim 21 , wherein the 2 nd HPLC pump is configured to pump the ethanol solution through the ethanol feed tube at a flow rate of about 50 mL/min to about 150 mL/min.

29 . The apparatus of claim 21 , wherein the ethanol solution is pumped at one third the flow rate of the aqueous solution.

29 . The apparatus of claim 21 , wherein the output solution has a flow rate of at least 200 mL/min, at least 300 mL/min, or at least 600 mL/min.

30 . The apparatus of claim 21 , wherein the output solution has a flow rate of about 200 mL/min to about 600 mL/min.

31 . The apparatus of claim 21 , wherein the apparatus is configured to produce an output solution flowing in the 1 st tube comprising a turbulent flow of the RNA and the one or more lipids is between about 10% to 75% ethanol v/v.

32 . The apparatus of claim 21 , wherein the RNA is encapsulated at greater than 98%.

33 . The apparatus of claim 21 , wherein the 1 st HPLC pump is configured to pump the aqueous solution through the 1 st tube with a back pressure of at least 10 psi, 25 psi, 50 psi, 75 psi, or 100 psi and the 2 nd HPLC pump is configured to pump the ethanol solution through the b 2 nd tube with a back pressure of at least 40 psi, 80 psi, 150 psi, 300 psi, or 400 psi.

34 . The apparatus of claim 21 , 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″.

35 . The apparatus of claim 21 , wherein the apparatus is configured to maintain the aqueous, ethanol, and output solutions at a temperature of about 15-20° C.

36 . The apparatus of claim 21 further comprising a 3 rd tube connected to a 3 rd HPLC pump configured for pumping a dilution buffer 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.

37 . The apparatus of claim 35 , wherein the 3 rd HPLC pump is configured to pump the dilution buffer through the 3 rd tube at a flow rate of 400-900 mL/min.

38 . The apparatus of claim 35 , wherein the 3 rd tube has an ID of 0.25″.

39 . The apparatus of claim 21 , wherein the lipid-encapsulated RNA nanoparticle composition has a polydispersity index of less than 0.09.

40 . The apparatus of claim 21 , wherein the lipid-encapsulated RNA nanoparticle composition has an average particle size of less than about 100 nm.