Rna formulations

Abstract

This disclosure provides improved lipid-based compositions, including lipid nanoparticle compositions, and methods of use thereof for delivering agents in vivo including nucleic acids and proteins.

1 . A composition comprising an enriched population of lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and optionally a structural lipid, wherein at least 50% of the LNPs comprise RNA encapsulated within the inner core and wherein the outer shell comprises at least 95% of the total PEG lipid in the LNP. 
     
     
         2 . A composition comprising an enriched population of lipid nanoparticles (LNPs) comprising RNA, wherein the LNPs have an outer shell and an inner core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and optionally a structural lipid, wherein at least 50% of the RNA in the composition is encapsulated within the LNPs and wherein the outer shell of the LNPs comprises at least 95% of the total PEG lipid in the LNP. 
     
     
         3 . A composition comprising an enriched population of lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid, wherein at least 50% of the LNPs have an outer shell fluidity value of greater than a threshold polarization level and wherein RNA is encapsulated within the LNP and wherein the outer shell comprises at least 95% of the total PEG lipid in the LNP. 
     
     
         4 . A composition comprising an enriched population of lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid, wherein at least 50% of the LNPs have a ratio of ionizable lipid:phospholipid:structural lipid of 35-50:30-40: 20-30, and wherein the PEG-lipid is a rapidly diffusing PEG-lipid. 
     
     
         5 . The composition of any one of  claims 1 - 4 , wherein a quantitative value of the amount of RNA encapsulated in the LNP is generated using an ion-exchange (IEX) chromatography assay. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the LNP is insensitive to accelerated blood clearance upon repeated administration in vivo within 10 days. 
     
     
         7 . The composition of any one of  claims 1 - 5 , wherein at least 50% of the LNPs have 1-5 inner shells. 
     
     
         8 . The composition of any one of  claims 1 - 5 , wherein the structural lipid is cholesterol. 
     
     
         9 . The composition of any one of  claims 1 - 5 , wherein 10-30% of the LNP, exclusive of RNA, is the structural lipid. 
     
     
         10 . The composition of any one of  claims 1 - 5 , wherein the outer shell is comprised of 10-30% of the structural lipid. 
     
     
         11 . The composition of any one of  claims 1 - 10 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs in the population have mRNA encapsulated therein, as determined by ion-exchange chromatography (TEX). 
     
     
         12 . The composition of any one of c s 1-11, wherein the LNPs have an encapsulation efficiency of at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, as determined by ion-exchange chromatography (TEX). 
     
     
         13 . A composition comprising a population of lipid nanoparticles (LNPs), the LNPs comprise an ionizable amino lipid, a poly(ethylene glycol) (PEG) lipid, a phospholipid, and optionally, a structural lipid, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs in the population have mRNA encapsulated therein, as determined by ion-exchange chromatography (TEX). 
     
     
         14 . A composition comprising a population of lipid nanoparticles (LNPs), the LNPs comprise an ionizable cationic lipid, a polyethylene glycol) (PEG) lipid, a phospholipid, and optionally, a structural lipid, wherein the LNPs have an encapsulation efficiency of at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, as determined by ion-exchange chromatography (IEX). 
     
     
         15 . The composition of  claim 13  or  14 , wherein the structural lipid is cholesterol or a cholesterol derivative. 
     
     
         16 . The composition of any of  claims 13 - 15 , wherein the composition is enriched for LNPs
 (a) lacking B1a cell-stimulating phospholipid epitopes, and/or   (b) lacking scavenger receptor ligands.   
     
     
         17 . The composition of  claim 16 , wherein at least about 50%, at east about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs
 (a) lack B1a cell-stimulating phospholipid epitopes, and/or   (b) lack scavenger receptor ligands.   
     
     
         18 . The composition of any of  claims 13 - 17 , wherein the composition is enriched for LNPs having a majority of the total phospholipid present in the outer LNP shell. 
     
     
         19 . The composition of  claim 18 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs having the majority of the total phospholipid present in the outer LNP shell. 
     
     
         20 . The composition of any of  claims 13 - 19 , wherein the composition is enriched for LNPs having more than 50% of the total phospholipid present on the surface (e.g., in outer LNP layers). 
     
     
         21 . The composition of  claim 20 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs have more than 50% of the total phospholipid present on the surface. 
     
     
         22 . The composition of any of  claims 13 - 21 , wherein the composition is enriched for LNPs having a majority of the total PEG lipid present in the outer LNP shell. 
     
     
         23 . The composition of  claim 22 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs having the majority of the total PEG lipid present in the outer LNP shell. 
     
     
         24 . The composition of any of  claims 13 - 23 , wherein the composition is enriched for LNPs having more than 50% of the total PEG lipid present on the surface (e.g., in outer LNP layers). 
     
     
         25 . The composition of  claim 25 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the LNPs have more than 50% of the total PEG lipid present on the surface. 
     
     
         26 . A composition, comprising:
 lipid nanoparticles (LNPs) cot comprising an ionizable lipid, a PEG lipid, and inaccessible mRNA; and   accessible mRNA, wherein no more than about 50% of mRNA in the composition is accessible mRNA, and wherein a half-life time of the PEG lipid in serum is less than or equal to about 3.0 hours.   
     
     
         27 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and one or more interior regions, wherein at least about 60% of the mRNA is positioned within the one or more interior regions and wherein at least about 60% of the PEG lipid is positioned within the exterior region.   
     
     
         28 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein at least about 50% of the PEG lipid in the lipid nanoparticles is surface accessible, and wherein no more than about 50% of mRNA in the composition is accessible mRNA.   
     
     
         29 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein a half-life time of the PEG lipid in serum is less than or equal to about 3.0 hours, and wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0,6.   
     
     
         30 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein a half-life of the PEG lipid in serum is less than or equal to about 3.0 hours, and wherein a surface polarity of the lipid nanoparticles is less than a threshold.   
     
     
         31 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein greater than about 50% of the PEG lipid in the lipid nanoparticles is surface accessible, and wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.6.   
     
     
         32 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein greater than about 50% of the PEG lipid in the lipid nanoparticles is surface accessible, and wherein a surface polarity of the lipid nanoparticles is less than a threshold.   
     
     
         33 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and one or more interior regions, wherein at least about 60% of the PEG lipid is positioned within the exterior region, and wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.5.   
     
     
         34 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and one or more interior regions, wherein at least about 60% of the PEG lipid is positioned within the exterior region, and wherein a surface polarity of the lipid nanoparticles is less than a threshold.   
     
     
         35 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid and inaccessible mRNA; and   accessible mRNA, wherein no more than about 50% of mRNA in the composition is accessible mRNA, and wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.5.   
     
     
         36 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid and inaccessible mRNA; and   accessible mRNA, wherein no more than about 50% of mRNA in the composition is accessible mRNA, and wherein a surface polarity of the lipid nanoparticles is less than a threshold.   
     
     
         37 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid and mRNA and having an exterior region and one or more interior regions, wherein at least about 60% of the mRNA is positioned within the one or more interior regions, and wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about pas.   
     
     
         38 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid and mRNA and having an exterior region and one or more interior regions, wherein at least about 60% of the MRNA is positioned within the one or more interior regions, and wherein a surface polarity of the lipid nanoparticles is less than a threshold.   
     
     
         39 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid and inaccessible mRNA; and   accessible mRNA, wherein no more than about 30% of mRNA in the composition is accessible mRNA.   
     
     
         40 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein at least about 50% of the lipid nanoparticles in the composition are enhanced lipid nanoparticles, and wherein the enhanced lipid nanoparticles have more inaccessible mRNA than the accessible mRNA.   
     
     
         41 . The composition of any preceding claim, wherein the PEG lipid comprising two or more aliphatic groups that are indirectly attached. 
     
     
         42 . The composition of any preceding claim, wherein a half-life time of the PEG lipid is greater than or equal to about 0.5 hours in the presence of serum. 
     
     
         43 . The composition of any preceding claim, wherein no more than about 30% of mRNA in the composition is accessible mRNA. 
     
     
         44 . The composition of any preceding claim, wherein a quantitative value of the amount of accessible mRNA is generated using an ion-exchange (LEX) chromatography assay. 
     
     
         45 . The composition of any preceding claim, wherein a quantitative value of the amount of accessible mRNA is not generated using a Ribogreen assay. 
     
     
         46 . The composition of any preceding claim, wherein no more than about 40%, no more than about 30%, no more than about 20%, or no more than about 10% of mRNA in the composition is accessible mRNA. 
     
     
         47 . The composition of any preceding claim, wherein at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the mRNA in the composition is fully encapsulated. 
     
     
         48 . The composition of any preceding claim, wherein a quantitative value of an amount of fully encapsulated mRNA is generated using an ion-exchange chromatography (TEX) assay. 
     
     
         49 . The composition of any preceding claim, wherein at least about 70%, at least about 80%, or at least about 90% of the mRNA is positioned within the one or more interior regions. 
     
     
         50 . The composition of any preceding claim, wherein at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the PEG lipid in the lipid nanoparticles is surface accessible. 
     
     
         51 . The composition of any preceding claim, wherein a half-life time of the PEG lipid in serum is less than or equal to about 2.75 hours, less than or equal to about 2.5 hours, or less than or equal to about 2 hours, less than or equal to about 2.5 hours, less than or equal to about 2.25 hours, less than or equal to about 2.0 hours, less than or equal to about 1.75 hours, less than or equal to about 1.5 hours, less than or equal to about 1.25 hours, less than or equal to about 1.0 hours, less than or equal to about 0.75 hours, less than or equal to about 0.5 hours, or less than or equal to about 0.25. 
     
     
         52 . The composition of any preceding claim, wherein at least about 70%, at least about 80%, or at least about 90% of the PEG lipid is positioned within the exterior region. 
     
     
         53 . The composition of any preceding claim, further comprising a continuous phase. 
     
     
         54 . The composition of any preceding claim, wherein the exterior region is in direct contact with the continuous phase. 
     
     
         55 . The composition of any preceding claim, (herein at least about 95% of the PEG lipid in the composition is surface accessible. 
     
     
         56 . The composition of any preceding claim, wherein at least about 95% of the PEG lipid is surface accessible in at least about 95% of the LNPs in the composition. 
     
     
         57 . The composition of any preceding claim, herein the surface polarity is determined using one or more fluorescent probes. 
     
     
         58 . The composition of any preceding claim, wherein the one or more fluorescent probes comprises prodan. 
     
     
         59 . The composition of any preceding claim, wherein the one or more fluorescent probes comprises laurdan. 
     
     
         60 . The composition of any preceding claim, wherein the threshold is the surface polarity of comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the comparative lipid nanoparticles comprise the same ionizable lipid, PEG lipid, and mRNA as the lipid nanoparticles, and wherein greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or 100% of the PEG lipid nanoparticles in the comparative lipid nanoparticles originated from the nanoprecipitation reaction. 
     
     
         61 . The composition of any preceding claim, wherein a normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.55, greater than or equal to about 0.60, greater than or equal to about 0.65, greater than or equal to about 0.70, greater than or equal to about 0.75, greater than or equal to about 0.80, or greater than or equal to about 0.85. 
     
     
         62 . The composition of any preceding claim, wherein the normalized general polarization of laurdan in the lipid nanoparticles is less than or equal to about 0.9. 
     
     
         63 . The composition of any preceding claim, wherein no more than about 5% of mRNA in the composition is accessible mRNA. 
     
     
         64 . The composition of any preceding claim, wherein an in vitro expression of the mRNA is greater than a threshold value. 
     
     
         65 . The composition of any preceding claim, wherein an in vitro expression of the mRNA is greater than comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the comparative lipid nanoparticles comprise the same ionizable lipid PEG lipid, and mRNA as the lipid nanoparticles, and wherein greater than 30% of the PEG lipid nanoparticles in the comparative lipid nanoparticles originated from the nanoprecipitation reaction. 
     
     
         66 . The composition of any preceding claim, wherein an in vivo expression of the mRNA is greater than a threshold value. 
     
     
         67 . The composition of any preceding claim, wherein an in vivo expression of the mRNA is greater than comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the comparative lipid nanoparticles comprise the same ionizable lipid, PEG lipid, and mRNA as the lipid nanoparticles, and wherein greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or 100% of the PEG lipid nanoparticles in the comparative lipid nanoparticles originated from the nanoprecipitation reaction. 
     
     
         68 . The composition of any preceding claim, wherein the PEG lipid is not a hydroxyl-PEG lipid. 
     
     
         69 . The composition of any preceding claim, wherein the PEG lipid is a methoxy-PEG lipid. 
     
     
         70 . The composition of any preceding claim, wherein the PEG lipid does not have the following structure: 
       
         
           
           
               
               
           
         
         wherein r is 45. 
       
     
     
         71 . The composition of any preceding claim, wherein the PEG-lipid is not Compounds 419, 420, 421, 422, 423, 424, 425, 426, 427, or 428. 
     
     
         72 . The composition of any preceding claim, wherein the lipid nanoparticles comprise a structural lipid. 
     
     
         73 . The composition of any preceding claim, wherein the lipid nanoparticles comprise a neutral lipid. 
     
     
         74 . The composition of any preceding claim, wherein the ionizable lipid is an ionizable amino lipid. 
     
     
         75 . The composition of any preceding claim, wherein the LNP has a molar ratio of ionizable amino lipid: structural lipid: neutral lipid: PEG-lipid other than 50:38.5:10:1.5. 
     
     
         76 . The composition of any preceding claim, wherein the PEG lipid is less than 1.5 in the molar ratio of ionizable amino lipid: structural lipid: neutral lipid: PEG-lipid. 
     
     
         77 . A composition, comprising:
 lipid nanoparticles (LNPs) comprising an ionizable lipid, a PEG lipid, and mRNA, wherein at least about 50% of the lipid nanoparticles in the composition are precursor lipid nanoparticles, the precursor lipid nanoparticles have more mRNA associated with the ionizable lipid than the PEG lipid, and the precursor lipid nanoparticles comprise at least about 0.01 mol % and less than or equal to about 1.0 mol % of the PEG lipid.   
     
     
         78 . The composition of any preceding claim, wherein at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the mRNA in the precursor lipid nanoparticles is associated with the ionizable lipid. 
     
     
         79 . The composition of any preceding claim, wherein less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the mRNA in the precursor lipid nanoparticles is associated with the PEG lipid. 
     
     
         80 . The composition of any preceding claim, wherein a ratio of mRNA associated with the ionizable lipid to mRNA associated with the PEG lipid in the precursor lipid nanoparticles is at least about 2:1, at least about 3:1, at least about 4:1, or at least about 5:1. 
     
     
         81 . The composition of any preceding claim, wherein the composition further comprises an organic solvent. 
     
     
         82 . The composition of any preceding claim, wherein the organic solvent comprises ethanol. 
     
     
         83 . The composition of any preceding claim, the precursor lipid nanoparticles comprise at least about 0.05 mol %, at least about 0.1 mol %, at least about 0.2 mol %, at least about 0.3 mol %, at least about 0.4 mol %, at least about 0.5 mol %, at least about 0.6 mol %, at least about 0.7 mol %, or at least about 0.8 mol % of the PEG lipid.