Methods for therapeutic administration of messenger ribonucleic acid drugs
The disclosure features methods of reducing or inhibiting an anti-drug antibody response in a subject, as well as methods of reducing or inhibiting unwanted immune cell activation in a subject to be treated with a messenger RNA (mRNA), comprising administering to the subject a mRNA, e.g., a chemically modified messenger RNA (mmRNA), encoding a polypeptide of interest, wherein the mRNA comprises at least one microRNA (miR) binding site for a miR expressed in immune cells, such as miR-126 binding site and/or miR-142 binding site, such that an anti-drug antibody response to the polypeptide or interest, or unwanted immune cell activation (e.g., B cell activation, cytokine secretion), is reduced or inhibited in the subject. The disclosure further provides therapeutic treatment regimens designed to reduce or inhibit ADA or unwanted immune cell activation (e.g., B cell activation, cytokine secretion) in a subject being treated with mRNA-based therapeutics.
1. A messenger RNA (mRNA) encoding a polypeptide of interest, wherein the mRNA comprises a 3′ untranslated region (UTR) comprising at least one miR-142 binding site and at least one miR-126 binding site, and wherein the mRNA comprises one or more modified nucleobases.
2. The mRNA of claim 1 , wherein the 3′UTR comprises 2, 3, or 4 miR-142 binding sites.
3. The mRNA of claim 1 , wherein the 3′UTR comprises 2, 3, or 4 miR-126 binding sites.
4. The mRNA of claim 1 , wherein the at least one miR-142 binding site is a miR-142-3p binding site.
5. The mRNA of claim 1 , wherein the at least one miR-142 binding site is a miR-142-5p binding site.
6. The mRNA of claim 1 , wherein the at least one miR-126 binding site a miR-126-3p binding site.
7. The mRNA of claim 1 , wherein the at least one miR-126 binding site is a miR-126-5p binding site.
8. The mRNA of claim 1 , wherein the one or more modified nucleobases is selected from pseudouridine (ψ), pseudouridine (ψ) and 5-methyl-cytidine (m 5 C), 1-methyl-pseudouridine (m 1 ψ), 1-methyl-pseudouridine (m 1 ψ) and 5-methyl-cytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine and 5-methyl-cytidine (m 5 C), 5-methoxy-uridine (mo 5 U), 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C), 2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine (m 5 C), N6-methyl-adenosine (m 6 A) or N6-methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C).
9. The mRNA of claim 1 , wherein all uracil nucleobases in the mRNA are modified uracil nucleobases.
10. The mRNA of claim 9 , wherein the modified uracil nucleobases are selected from pseudouridines (ψ), 1-methyl-pseudouridines (m 1 ψ), and 5-methoxy-uridines (mo 5 U).
11. The mRNA of claim 9 , wherein the modified uracil nucleobases are 1-methyl-pseudouridines (m 1 ψ).
12. The mRNA of claim 1 , wherein all cytosine nucleobases in the mRNA are modified cytosine nucleobases.
13. The mRNA of claim 12 , wherein the modified cytosine nucleobases are 5-methyl-cytidine.
14. The mRNA of claim 1 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or fusion protein.
15. A mRNA encoding a polypeptide of interest, wherein the mRNA comprises a 3′ UTR comprising at least one miR-142-3p binding site and at least one miR-126-3p binding site, and wherein the mRNA comprises one or more modified nucleobases.
16. The mRNA of claim 15 , wherein the 3′UTR comprises 2, 3, or 4 miR-142-3p binding sites.
17. The mRNA of claim 15 , wherein the 3′UTR comprises 2, 3, or 4 miR-126-3p binding sites.
18. The mRNA of claim 15 , wherein the miR-142-3p binding site comprises the sequence of SEQ ID NO: 3.
19. The mRNA of claim 15 , wherein the miR-126-3p binding site comprises the sequence of SEQ ID NO: 26.
20. The mRNA of claim 15 , wherein the one or more modified nucleobases is selected from pseudouridine (ψ), pseudouridine (ψ) and 5-methyl-cytidine (m 5 C), 1-methyl-pseudouridine (m 1 ψ), 1-methyl-pseudouridine (m 1 ψ) and 5-methyl-cytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine and 5-methyl-cytidine (m 5 C), 5-methoxy-uridine (mo 5 U), 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C), 2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine (m 5 C), N6-methyl-adenosine (m 6 A) or N6-methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C).
21. The mRNA of claim 15 , wherein all uracil nucleobases in the mRNA are modified uracil nucleobases.
22. The mRNA of claim 21 , wherein the modified uracil nucleobases are selected from pseudouridines (ψ), 1-methyl-pseudouridines (m 1 ψ), and 5-methoxy-uridines (mo 5 U).
23. The mRNA of claim 21 , wherein the modified uracil nucleobases are 1-methyl-pseudouridines (m 1 ψ).
24. The mRNA of claim 15 , wherein all cytosine nucleobases in the mRNA are modified cytosine nucleobases.
25. The mRNA of claim 24 , wherein the modified cytosine nucleobases are 5-methyl-cytidine.
26. The mRNA of claim 15 , wherein the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody or fusion protein.
27. A lipid nanoparticle comprising the mRNA of claim 1 , wherein the lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and a PEG lipid.
28. A pharmaceutical composition comprising the lipid nanoparticle of claim 27 and a pharmaceutically acceptable carrier, diluent or excipient.
29. A lipid nanoparticle comprising the mRNA of claim 15 , wherein the lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and a PEG lipid.
30. A pharmaceutical composition comprising the lipid nanoparticle of claim 29 and a pharmaceutically acceptable carrier, diluent or excipient.
31. The mRNA of claim 15 , wherein the 3′UTR comprises one miR-142-3p binding site comprising the sequence of SEQ ID NO: 3 and one miR-126-3p binding site comprising the sequence of SEQ ID NO: 26.
32. The mRNA of claim 31 , wherein all uracil nucleobases in the mRNA are modified uracil nucleobases, and wherein the modified uracil nucleobases are 1-methyl-pseudouridines (m1ψ).