Lipid formulations for delivery of messenger RNA

Patent metadata

Field	Value
Publication number	US-10959953-B2
Application number	US-201916685287-A
Country	US
Kind code	B2
Filing date	Nov 15, 2019
Priority date	Oct 22, 2013
Publication date	Mar 30, 2021
Grant date	Mar 30, 2021

Abstract

The present invention provides, among other things, methods of delivering mRNA in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a 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.

First claim

We claim:

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 protein that is argininosuccinate synthetase (ASS1), 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 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 C1-50 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-dieleeyl-(1,2-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 a claim 3 , wherein the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

7. The method of 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 composition is administered intravenously.

9. The method of claim 1 , wherein the expression of the argininosuccinate synthetase (ASS1) protein encoded by the mRNA is detectable in liver.

10. The method of claim 1 , wherein the mRNA comprises one or more modified nucleotides that are 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-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and/or 2-thiocytidine.

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

12. A method of treating citrullinemia, type I comprising
administering to subject in need of treatment a composition comprising an mRNA encoding a therapeutic protein that is argininosuccinate synthetase (ASS1) encapsulated within a liposome such that the administering of the composition results in the expression of the ASS1 protein encoded by the mRNA in one or more tissues affected by the citrullinemia, type I;

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 C1-50 alkyl, optionally substituted C2-50 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.

13. The method of claim 12 , wherein the cationic lipid is cKK-E12:

14. A composition for delivery of messenger RNA (mRNA) comprising an mRNA encoding a protein that is argininosuccinate synthetase (ASS1) 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 C 1-6 alkyl;
each instance of R 6 and R 7 is independently a group of the

Assignees

• Translate Bio Inc
• Massachusetts Inst Technology

Inventors

• Heartlein Michael
• Anderson Daniel
• Dong Yizhou
• Derosa Frank

Classifications

• A61K9/1272 Primary

  comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543}) · CPC title

• A61K9/12

  Aerosols; Foams {(A61K9/0043, A61K9/0056, A61K9/006, A61K9/0073 take precedence; spray-films A61K9/7015)} · CPC title

• A61K9/0073

  Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy; (nasal sprays A61K9/0043; inhalation of vapours of volatile or heated drugs, e.g. essential oils or nicotine, A61K9/007; devices A61M) · CPC title

• A61K9/0019 Primary

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title