ENCAPSULATION OF MESSENGER RNA

• US 20170348244A1
• Filed: 04/24/2017
• Published: 12/07/2017
• Est. Priority Date: 07/02/2014
• Status: Abandoned Application

• Current Assignee: Translate Bio, Inc. (Sanofi )
• Original Assignee: RaNA Therapeutics Inc. (Sanofi )
• Inventors: DeRosa, Frank, Karve, Shrirang, Heartlein, Michael
• Application Number:
  US15/495,157
• Publication Number:
  US 20170348244A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: A61K 9/5015 Organic compounds, e.g. fats, sugars
• CPC Class Codes: A61K 9/5089 Processes

Abstract

The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.

Claims

1. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.

2. The process of claim 1, wherein the pre-determined temperature is or is greater than about 30°

C., 37°

C., 40°

C., 45°

C., 50°

C., 55°

C., 60°

C., 65°

C., or 70°

C.

3. The process of claim 1, wherein the pre-determined temperature ranges from about 25-70°

C., about 30-70°

C., about 35-70°

C., about 40-70°

C., about 45-70°

C., about 50-70°

C., or about 60-70°

C.

5. The process of claim 1, wherein the mRNA solution and the lipid solution are heated to the pre-determined temperature separately prior to the mixing.

9. The process of claim 1, wherein the mRNA solution and the lipid solution are mixed by a pulse-less flow pump.

10. The process of claim 9, wherein the pump is a gear pump.

11. The process of claim 9, wherein the pump is a centrifugal pump.

12. The process of claim 1, wherein the mRNA solution is mixed at a flow rate ranging from about 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute.

14. The process of claim 1, wherein the lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, about 75-200 ml/minute, about 200-350 ml/minute, about 350-500 ml/minute, about 500-650 ml/minute, about 650-850 ml/minute, or about 850-1000 ml/minute.

23. The process of claim 1, wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol.

24. The process of claim 1, wherein the mRNA solution and the lipid solution are mixed into a 20% ethanol, resulting in a suspension of lipid nanoparticles.

25. The process of claim 24, wherein the lipid nanoparticles are further purified by Tangential Flow Filtration.

26. The process of claim 25, wherein greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles have a size less than 100 nm.

30. The process of claim 25, wherein the purified nanoparticles have an encapsulation rate of greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

42. The process of claim 1, wherein the one or more cationic lipids are selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

43. The process of claim 1, wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′

-rac-glycerol)).

4. (canceled)

6-8. -8. (canceled)

13. (canceled)

15-22. -22. (canceled)

27-29. -29. (canceled)

31. (canceled)

32. A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising

a. Separately heating a mRNA solution and/or a lipid solution to a pre-determined temperature greater than ambient temperature;

b. Mixing the heated mRNA solution and/or the heated lipid solution to generate a suspension of lipid nanoparticles; and

c. Purifying the lipid nanoparticles.

34. The process of claim 32, wherein a composition of lipid nanoparticles is generated.

46. The process of claim 32, wherein the mRNA comprises one or more modified nucleotides.

35. A composition comprising purified lipid nanoparticles, wherein greater than about 90% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm and greater than about 70% of the purified lipid nanoparticles encapsulate a mRNA within each individual particle.

36-41. -41. (canceled)

44-45. -45. (canceled)

47. (canceled)