Lipid nanoparticles encapsulation of large rna

Patent metadata

Field	Value
Publication number	US-2022168234-A1
Application number	US-202117473063-A
Country	US
Kind code	A1
Filing date	Sep 13, 2021
Priority date	Sep 13, 2020
Publication date	Jun 2, 2022
Grant date	—

Abstract

A method of producing lipid-encapsulated RNA nanoparticles includes flowing an aqueous solution comprising an RNA through a 1 st tube having a first inner diameter (ID); the RNA comprises from about 6,000 to about 13,000 nucleotides; flowing an ethanol solution comprising lipids through a 2 nd tube having a second inner diameter (ID), at a flow rate of about 0.2 to about 1 times relative to the aqueous solution through the 1 st tube, the lipids comprise a cationic lipid; and mixing the ethanol solution with the aqueous solution; the first ID and second ID and flow rates through the 1 st tube and 2 nd tube are selected to produce a shear force sufficiently low to preserve the integrity of the RNA; 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 ethanol, the lipid-encapsulated RNA nanoparticles having a bilayer structure.

First claim

1 . A method of producing a lipid-encapsulated RNA nanoparticle, comprising the steps
a) flowing an aqueous solution comprising an RNA through a 1s t tube having an inner diameter (ID) of from about 0.01 inches to about 0.08 inches; wherein a pH of the aqueous solution is in a range from about 3.0 to about 4.5 with an optional NaCl concentration of up to about 300 mM;
wherein the RNA comprises from about 6,000 to about 13,000 nucleotides;
b) flowing an ethanol solution comprising lipids through a 2 nd tube having an ID of from about 0.01 inches to about 0.04 inches at a flow rate of about 0.2 to about 1 times a 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;
wherein the mixing produces an output solution flowing in the 1 st tube comprising a turbulent flow of the RNA and the lipids in about 10% to 75% ethanol v/v; and
wherein the lipid-encapsulated RNA nanoparticles have a bilayer structure.

2 . 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 a first inner diameter (ID);
wherein the RNA comprises from about 6,000 to about 13,000 nucleotides;
b) flowing an ethanol solution comprising lipids through a 2 nd tube having a second inner diameter (ID), at a flow rate of about 0.2 to about 1 times a 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;
wherein the first ID and second ID and flow rates through the 1 st tube and 2 nd tube are selected to produce a shear force sufficiently low to preserve the integrity of the RNA;
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; and

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

3 . The method of claim 1 , wherein the mixing comprises 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.

4 . The method of claim 1 , wherein the mixing comprises flowing the ethanol solution and the aqueous solution into a multi-inlet vortex mixer.

5 . The method of claim 1 , wherein a concentration of RNA in the aqueous solution is in a range from about 85 micrograms/mL to about 2100 micrograms/mL.

6 . The method of claim 1 , wherein a concentration of lipid in the ethanol solution is in a range from about 5.0 mg/mL to about 125 mg/mL.

7 . The method of claim 1 , wherein the aqueous solution is pumped through the 1 st tube by a 1 st pump with a back pressure of not more than about 200 psi, and the ethanol solution is pumped through the 2 nd tube by a 2 nd pump.

8 . The method of claim 1 , wherein the 1 st tube has an ID in a range from about 0.02 inches to about 0.03 inches and the 2 nd tube has an ID in a range from about 0.01 inches to about 0.02 inches.

9 .- 12 . (canceled)

13 . The method of claim 1 , wherein the aqueous solution is pumped at a flow rate in a range from about 40 mL/min. to about 375 mL/min.

14 .- 15 . (canceled)

16 . The method of claim 1 , wherein the aqueous, ethanol, and output solutions are maintained in a temperature range from about 10° C. to about 25° C.

17 . The method of claim 1 , further comprising pumping a first dilution buffer and mixing the dilution buffer with the output solution by introducing the dilution buffer to the output solution to produce a first diluted output solution.

18 . The method of claim 17 , further comprising pumping a second dilution buffer into the first diluted output solution thereby forming a final diluted output solution, wherein there is a delay between pumping the first dilution buffer and second dilution buffer.

19 . (canceled)

20 . The method of claim 17 , wherein the first dilution buffer comprises:
a) a buffering agent having a pH from about 5.5 to about 7.0; and b) optionally a sodium chloride concentration up to about 100 mM; and the second dilution buffer comprises: a) a buffering agent having a pH between about 7.4 and 8.0; and b) optionally a sodium chloride concentration up to about 100 mM.

21 . (canceled)

22 . The method of claim 18 , wherein the second buffer comprises sucrose up to about 15% w/v, an antioxidant up to about 0.5% w/v, up to 20 mM of a chelating agent, or any combination of the foregoing.

23 .- 24 . (canceled)

25 . The method of claim 17 , wherein the first diluted output solution comprises about 1.0% to about 10.0% ethanol.

26 . The method of claim 17 , wherein the first dilution buffer is pumped at a flow rate from about 80 mL/min. to about 900 mL/min and the second dilution buffer is pumped at a flow rate from about 240 mL/min to about 5400 mL/min.

27 .- 28 . (canceled)

29 . The method of claim 1 , wherein the lipid-encapsulated RNA nanoparticle has an average particle size in a range from about 50 nm to about 120 nm.

30 .- 31 . (canceled)

32 . The method of claim 1 , wherein the lipid portion of the lipid-encapsulated RNA nanoparticle further comprises one or more agents selected from the group consisting of a helper lipid, a cholesterol, and a PEG lipid conjugate.

33 . The method of claim 1 , wherein the RNA is self-replicating RNA.

34 . The method of claim 1 , further comprising lyophilizing the final diluted output solution.

Assignees

• Arcturus Therapeutics Inc

Inventors

• Bao Yanjie
• Clemente Brenda
• Karmali Priya Prakash

Classifications

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2320/32

  Special delivery means, e.g. tissue-specific · CPC title

• A61K9/5192 Primary

  Processes · CPC title

• A61K9/5123

  Organic compounds, e.g. fats, sugars · CPC title