Polynucleotides encoding interleukin-12 (IL12) and uses thereof
The present disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human interleukin-12 (IL12), functional fragments thereof, and fusion proteins comprising IL12. In some embodiments, the open reading frame is sequence-optimized. In particular embodiments, the disclosure provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human IL12, or sequences having high sequence identity with those sequence optimized polynucleotides.
1. A method for treating cancer in a subject by inducing an anti-tumor immune response, comprising administering intratumorally to the subject a lipid nanoparticle (LNP) encapsulated messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a human IL-12 polypeptide, wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid; 5-25% phospholipid; 25-55% sterol; and 0.5-15% PEG-modified lipid.
2. The method of claim 1 , wherein the ORF encodes a human IL-12B polypeptide operably linked to a human IL-12A polypeptide.
3. The method of claim 2 , wherein the IL-12B polypeptide is operably linked to the IL-12A polypeptide by a peptide linker, and wherein the peptide linker comprises a Gly/Ser linker.
4. The method of claim 1 , wherein the human IL12 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 48.
5. The method of claim 1 , wherein the anti-tumor response comprises a T-cell response, IFNγ production, or both a T-cell response and IFNγ production.
6. A method for treating cancer in a subject by inducing an anti-tumor immune response, comprising administering intratumorally to the subject a LNP encapsulated mRNA comprising an ORF encoding a human IL-12 polypeptide, wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, and wherein the mRNA comprises an ORF comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 237.
7. The method of claim 6 , wherein the mRNA comprises the nucleotide sequence as set forth in SEQ ID NO: 237.
8. The method of claim 7 , wherein the mRNA comprises a 3′ UTR comprising a microRNA binding site, wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site.
9. The method of claim 8 , wherein the miR-122-5p binding site comprises the sequence set forth in SEQ ID NO: 54.
10. The method of claim 7 , wherein the mRNA comprises a 3′ UTR comprising the sequence set forth in SEQ ID NO: 240.
11. The method of claim 10 , wherein the mRNA comprises a 5′ UTR comprising the sequence set forth in SEQ ID NO: 135.
12. The method of claim 7 , wherein the mRNA is fully modified with chemically-modified uridines.
13. The method of claim 12 , wherein the chemically-modified uridines are N1-methylpseudouridines (m1ψ).
14. The method of claim 6 , wherein the anti-tumor response comprises a T-cell response, IFNγ production, or both a T-cell response and IFNγ production.
15. The method of claim 1 , wherein the mRNA comprises a nucleotide sequence at least 90% to SEQ ID NO: 96.
16. The method of claim 15 , wherein the mRNA comprises the nucleotide sequence as set forth in SEQ ID NO: 96.
17. The method of claim 16 , wherein the mRNA is fully modified with N1-methylpseudouridines (m1ψ).
18. The method of claim 1 , wherein the ionizable amino lipid comprises a compound having the formula
19. The method of claim 1 , wherein the LNP comprises a molar ratio of about 50% ionizable amino lipid; 10% phospholipid; 38.5% cholesterol; and 1.5% PEG-modified lipid.
20. The method of claim 19 , wherein the ionizable amino lipid comprises a compound having the formula