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 human survival of motor neuron-1 (hSMN-1) 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 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 1 , wherein the expression of the human survival of motor neuron-1 (hSMN-1) protein encoded by the mRNA is detectable in brain and cerebrospinal fluid.

5. 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.

6. A method of treating spinal muscular atrophy comprising:

administering to subject in need of treatment a composition comprising an mRNA encoding a human survival of motor neuron-1 (hSMN 1) protein encapsulated within a liposome such that the administering of the composition results in the expression of the hSMN-1 protein encoded by the mRNA in one or more tissues affected by spinal muscular atrophy;

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 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.

7. The method of claim 6 , wherein the cationic lipid is cKK-E12:

8. A composition for delivery of messenger RNA (mRNA) comprising an mRNA encoding a human survival of motor neuron-1 (hSMN-1) protein encapsulated within a liposome, 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 C1-6 alkyl; 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.

9. The composition of claim 8 , wherein the cationic lipid is cKK-E12:

10. The composition of claim 8 , 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.

11. The composition of claim 10 , wherein the cationic lipid constitutes about 30-50% of the liposome by molar ratio.

12. The composition of claim 8 , wherein the liposome has a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm.

13. The composition of claim 8 , wherein the composition is formulated for intrathecal administration.