Methods of making lipid nanoparticles
The disclosure features novel methods of producing nucleic acid lipid nanoparticle (LNP) compositions employing a modifying agent after formation of a precursor nucleic acid lipid nanoparticle, the produced compositions thereof, and methods involving the nucleic acid lipid nanoparticles useful in the delivery of therapeutics and/or prophylactics, such as a nucleic acid, to mammalian cells or organs to, for example, to regulate polypeptide, protein, or gene expression.
1. A method of producing a nucleic acid lipid nanoparticle composition, the method comprising:
mixing a lipid solution comprising an ionizable lipid with a solution comprising a nucleic acid thereby forming a precursor nucleic acid lipid nanoparticle, wherein the precursor nucleic acid lipid nanoparticle comprises a first PEG lipid;
optionally processing the precursor nucleic acid lipid nanoparticle;
adding a second PEG lipid to the precursor nucleic acid lipid nanoparticle thereby forming a modified nucleic acid lipid nanoparticle; and
processing the modified nucleic acid lipid nanoparticle, thereby forming the nucleic acid lipid nanoparticle composition,
wherein:
the first PEG lipid and the second PEG lipid are independently
i) a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, or a PEG-modified dialkylglycerol;
ii) a compound of Formula (PL-II):
or a salt thereof, wherein:
R 3 is —OR O ;
R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;
r is an integer between 1 and 100;
R 5 is optionally substituted C 10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R 5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O) N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(═NR N ), C(═NR N )N(R N ), NR N C(═NR N ), NR N C(═NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O) 2 , S(O) 2 O, OS(O) 2 O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O) 2 , N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or N(R N )S(O) 2 O; and
each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group, or
iii) a compound of Formula (PL-III):
wherein s is an integer between 1 and 100.
2. The method of claim 1 , wherein at least one of the first PEG lipid and the second PEG lipid is a compound of Formula (PL-II-OH):
or a salt thereof, wherein:
r is an integer between 1 and 100;
R 5 is optionally substituted C 10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R 5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, C(O)S, SC(O), C(═NR N ), C(═NR N )N(R N ), NR N C(═NR N ), NR N C(═NR N )N(R N ), C(S), C(S)N(R N ), NR N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O) 2 , S(O) 2 O, OS(O) 2 O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O) 2 , N(R N )S(O) 2 , S(O) 2 N(R N ), N(R N )S(O) 2 N(R N ), OS(O) 2 N(R N ), or N(R N )S(O) 2 O; and
each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.
3. The method of claim 1 , wherein at least one of the first PEG lipid and the second PEG lipid is a compound of Formula (PL-II), wherein the compound of Formula (PL-II) is:
4. The method of claim 1 , wherein at least one of the first PEG lipid and the second PEG lipid is a compound of Formula (PL-II), wherein the compound of Formula (PL-II) is:
5. The method of claim 1 , wherein at least one of the first PEG lipid and the second PEG lipid is a compound of following formula:
6. The method of claim 1 , wherein the molar ratio of the first PEG lipid to the second PEG lipid is in a range of about 1:100 to about 1:1, about 1:50 to about 1:1, about 1:25 to about 1:1, or about 1:10 to about 1:1.
7. The method of claim 1 , wherein the precursor nucleic acid lipid nanoparticle is processed prior to adding the second PEG lipid.
8. The method of claim 1 , wherein the precursor nucleic acid lipid nanoparticle is not processed prior to adding the second PEG lipid.
9. The method of claim 1 , wherein the mixing comprises turbulent mixing and/or microfluidic mixing.
10. The method of claim 1 , wherein processing the modified nucleic acid lipid nanoparticle comprises filtration, freezing and/or lyophilizing, and/or packing.
11. The method of claim 1 , wherein the precursor nucleic acid lipid nanoparticle further comprises a phospholipid and/or a structural lipid.
12. The method of claim 11 , wherein the nucleic acid lipid nanoparticle composition comprises about 30-60 mol % of the ionizable lipid;
about 0-30 mol % of the phospholipid;
about 15-50 mol % of the structural lipid; and
about 0.01-20 mol % total amount of the first PEG lipid and second PEG lipid.
13. The method of claim 1 , wherein the nucleic acid is a messenger RNA (mRNA).