Lipid formulations for delivery of messenger RNA

1. A method of delivery of messenger RNA (mRNA) in vivo, comprising administering to a subject in need of delivery a composition comprising an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo;

wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof,

wherein:

p is an integer of between 1 and 9, inclusive;

each instance of R 2 is independently hydrogen or optionally substituted C 1-6 alkyl;

each instance of L is independently an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, or combination thereof;

each instance of R 6 and R 7 is independently a group of the formula (i), (ii), or (iii);

Formulae (i), (ii), and (iii) are:

wherein:

each instance of R′ is independently hydrogen or optionally substituted alkyl;

X is O, S, or NR X , wherein R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

Y is O, S, or NR Y , wherein R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and

R L is optionally substituted C 1-50 alkyl, optionally substituted C 2-50 alkenyl, optionally substituted C 2-50 alkynyl, optionally substituted heteroC 1-50 alkyl, optionally substituted heteroC 2-50 alkenyl, optionally substituted heteroC 2-50 alkynyl, or a polymer.

2. The method of claim 1 , wherein the cationic lipid is cKK-E12:

3. The method of claim 1 , wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.

4. The method of claim 3 , 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)).

5. The method of claim 3 , wherein the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol.

6. The method of claim 3 , wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

7. The method claim 1 , wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.

8. The method of claim 1 , wherein the mRNA comprises one or more modified nucleotides, and wherein the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.

9. The method of claim 1 , wherein the mRNA is unmodified.

10. A method of delivery of messenger RNA (mRNA) in vivo, comprising administering to a subject in need of delivery a composition comprising an mRNA encoding a ornithine transcarbamylase (OTC) protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo;

wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof,

wherein:

p is an integer of between 1 and 9, inclusive;

each instance of R 2 is independently hydrogen or optionally substituted C 1-6 alkyl;

each instance of L is independently an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene, or combination thereof;

each instance of R 6 and R 7 is independently a group of the formula (i), (ii), or (iii);

Formulae (i), (ii), and (iii) are:

wherein:

each instance of R′ is independently hydrogen or optionally substituted alkyl;

X is O, S, or NR X , wherein R X is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

Y is O, S, or NR Y , wherein R Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

R P is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; and

R L is optionally substituted C 1-50 alkyl, optionally substituted C 2-50 alkenyl, optionally substituted C 2-50 alkynyl, optionally substituted heteroC 1-50 alkyl, optionally substituted heteroC 2-50 alkenyl, optionally substituted heteroC 2-50 alkynyl, or a polymer.

11. The method of claim 10 , wherein the cationic lipid is cKK-E12:

12. The method of claim 10 , wherein the liposome further comprises one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids.

13. The method of claim 12 , wherein the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phospho choline), 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)).

14. The method of claim 12 , wherein the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol.

15. The method of claim 12 , wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

16. The method claim 10 , wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.

17. The method of claim 10 , wherein the mRNA comprises one or more modified nucleotides, and wherein the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.

18. The method of claim 10 , wherein the mRNA is unmodified.