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 full length mRNA molecule that encodes a full length protein, having a coding region and optionally, one or more non-coding regions, wherein the mRNA is at least 500 nucleotide residues in length; at least 1% of the total mRNA nucleotide residues incorporate a 4′-thio-substituted furanose ring, and up to 20% of the total mRNA nucleotide residues incorporate a 4′-thio-substituted furanose ring, and wherein the mRNA, upon delivery in vivo, produces a protein encoded by the coding region.
2. The mRNA molecule of claim 1 , wherein at least 5% of the total mRNA nucleotide residues incorporate a 4′-thio-substituted furanose ring.
3. The mRNA molecule of claim 1 , wherein at least 10% of the total mRNA nucleotide residues incorporate a 4′-thio-substituted furanose ring.
4. The mRNA molecule of claim 1 , wherein the non-coding region comprises a poly-A tail, and wherein the poly-A tail comprises 4′-thio-adenosine residues.
5. The mRNA molecule of claim 4 , wherein the poly-A tail is at least 90 nucleotide residues in length.
6. The mRNA molecule of claim 1 , wherein the mRNA further comprises at least one nonstandard nucleotide residue.
7. The mRNA molecule of claim 1 , wherein the molecule comprises at least 5000 nucleotide residues.
8. The mRNA molecule of claim 1 , wherein the coding region encodes a full length protein that is a therapeutic protein.
9. The mRNA molecule of claim 8 , wherein the therapeutic protein is chosen from erythropoietin, human growth hormone, cystic fibrosis transmembrane conductance regulator (CFTR), insulin, alpha-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase, heparan sulfamidase, hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1), glucose-6-phosphatase, glucose-6-phosphate translocase, glycogen debranching enzyme, lysosomal alpha-glucosidase, 1,4-alpha-glucan branching enzyme, glycogen phosphorylase, phosphofructokinase, liver phosphorylase, GLUT-2, UDP glycogen synthase, alpha-L-iduronidase, iduronate sulfate silfatase, heparan sulfate sulfamidase, alpha-N-acetylglucose amidase, alpha-glucosaminid-N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, apolipoprotein E, low density lipoprotein receptor (LDLR), Factor VIII, Factor IX, spinal motor neuron 1 (SMN1), phenylalanine hydroxylase, propionyl-CoA carboxylase, porphobilinogen deaminase, pethylmalonyl-CoA mutase, urate oxidase, C1 esterase inhibitor, and acid alpha-glucosidase.
10. A composition comprising at least one mRNA molecule of claim 1 and a carrier that is a lipid nanoparticle or a polymer.
11. The composition of claim 10 , wherein the carrier is a lipid nanoparticle that comprises one or more cationic lipids selected from XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003, ICE, HGT5000, cis or trans HGT5001, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, and C12-200.
12. The composition of claim 10 , wherein the carrier is a lipid nanoparticle that comprises one or more helper lipids 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), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and cholesterol.
13. The composition of claim 10 , wherein the carrier is a lipid nanoparticle that comprises a pegylated lipid.
14. The composition of claim 10 , wherein the lipid nanoparticle comprises one or more cationic lipids, one or more helper lipids, and a pegylated lipid.
15. The composition of claim 10 , wherein the carrier comprises a polymer.
16. The composition of claim 15 , wherein the polymer is polyethyleneimine.
17. A method of producing a protein in vivo, comprising administering the mRNA of claim 1 to a subject.