ENCAPSULATION OF MESSENGER RNA

• US 20180263918A1
• Filed: 05/14/2018
• Published: 09/20/2018
• Est. Priority Date: 07/02/2014
• Status: Abandoned Application

• Current Assignee: Translate Bio, Inc. (Sanofi )
• Original Assignee: Translate Bio, Inc. (Sanofi )
• Inventors: DeRosa, Frank, Karve, Shrirang, Heartlein, Michael
• Application Number:
  US15/979,054
• Publication Number:
  US 20180263918A1
• 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

1-47. -47. (canceled)

48. A process of encapsulating messenger RNA (mRNA) encoding cystic fibrosis conductance regulator protein in lipid nanoparticles at an encapsulation efficiency of 85% or greater, the process comprising:

mixing by a pulse-less flow pump and at a pre-determined temperature greater than ambient temperature

(i) an mRNA solution comprising mRNA encoding cystic fibrosis conductance regulator protein and

(ii) a lipid solution comprising one or more cationic lipids, one or more helper lipids, and one or more PEG lipids,

to produce lipid nanoparticles encapsulating cystic fibrosis conductance regulator protein at an encapsulation efficiency of 85% or greater,

wherein the lipid nanoparticles encapsulating cystic fibrosis conductance regulator protein from the mixing have an average size of 80 nm or less.

49. The process of claim 48, wherein the mRNA solution comprising mRNA encoding cystic fibrosis conductance regulator protein is mixed at a flow rate that is faster than the flow rate of the lipid solution.

50. The process of claim 49, wherein the mRNA solution comprising mRNA encoding cystic fibrosis conductance regulator protein is mixed at a flow rate that is about four times faster than the flow rate of the lipid solution.

51. The process of claim 48, wherein the mRNA solution comprising mRNA encoding cystic fibrosis conductance regulator protein is in an acidic buffer at about pH 4.5.

52. The process of claim 48, wherein the mRNA solution and the lipid solution are mixed into a dilute ethanol, resulting in a suspension of lipid nanoparticles.

53. The process of claim 48, wherein the lipid nanoparticles are further purified by Tangential Flow Filtration.

54. A nanoparticle formulation comprising lipid nanoparticles encapsulating mRNA encoding cystic fibrosis conductance regulator protein,

wherein the mRNA encoding cystic fibrosis conductance regulator protein is encapsulated in the lipid nanoparticles at an encapsulation efficiency of 85% or greater, and

wherein the lipid nanoparticles have an average size of 80 nm or less.

55. The nanoparticle formulation of claim 54, wherein the lipid nanoparticles comprise one or more cationic lipids, one or more helper lipids, and one or more PEG lipids.

56. The nanoparticle formulation of claim 55, 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.

57. The nanoparticle formulation of claim 55, wherein the one or more helper 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)).

58. The nanoparticle formulation of claim 55, wherein the lipid nanoparticles further comprise one or more cholesterol-based lipids.