Ribonucleic acids with 4′-thio-modified nucleotides and related methods
Disclosed are messenger RNA molecules and related compositions incorporating a 4′-thio modification in the furanose ring of at least one nucleotide residue, and methods of using these mRNAs to produce an encoded therapeutic protein in vivo and to treat or prevent diseases or disorders. In certain embodiments, the 4′-thio modified mRNA provides for enhanced stability and/or reduced immunogenicity in in vivo therapies.
1. A lipid nanoparticle comprising:
at least one full-length mRNA molecule, wherein said full-length mRNA molecule has a coding region and, optionally, one or more non-coding regions,
encodes a full length protein, and
is at least 500 nucleotide residues in length, and
wherein 1%-20% of the total mRNA nucleotide residues of the full-length mRNA incorporate a 4′-thio-substituted furanose ring; and
wherein said lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid, and a PEGylated lipid; and
wherein said mRNA is encapsulated within said lipid nanoparticle.
2. The lipid nanoparticle of claim 1 , wherein the lipid nanoparticle comprises:
a non-cationic lipid selected from 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), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol));
and
a cholesterol-based lipid that is cholesterol.
3. A method of producing a protein in vivo, comprising administering the lipid nanoparticle of claim 1 to a subject.
4. The method of claim 3 , wherein the method produces a therapeutic protein in vivo.
5. A pharmaceutical composition comprising the lipid nanoparticle of claim 1 .
6. The lipid nanoparticle of claim 1 , wherein the mRNA further comprises at least one nonstandard nucleotide residue.
7. The lipid nanoparticle of claim 6 , wherein the mRNA further comprises at least one nonstandard nucleotide residue selected from the group consisting of: 5-methyl-cytidine (“5mC”), pseudouridine (“ψU”), 2-thio-uridine (“2sU”), 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine.
8. The lipid nanoparticle of claim 6 , wherein the mRNA further comprises at least one nonstandard nucleotide residue having a sugar modification that is a 2′-O-alkyl modification.
9. The lipid nanoparticle of claim 8 , wherein the 2′-O-alkyl modification is a 2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a 2′-O-methoxyethyl modification or a 2′-deoxy modification.