Polynucleotides encoding interleukin-12 (IL12) and uses thereof

Patent metadata

Field	Value
Publication number	US-11311602-B2
Application number	US-202117308686-A
Country	US
Kind code	B2
Filing date	May 5, 2021
Priority date	May 18, 2016
Publication date	Apr 26, 2022
Grant date	Apr 26, 2022

Abstract

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.

First claim

What is claimed is:

1. A lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, wherein the IL-12B polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the IL-12A polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid.

2. The LNP of claim 1 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide without a linker.

3. The LNP of claim 1 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide with a peptide linker.

4. The LNP of claim 1 , wherein the fusion protein comprises a heterologous signal peptide.

5. The LNP of claim 1 , wherein the fusion protein comprises a human IL-12B signal peptide or a human IL-12A signal peptide.

6. The LNP of claim 5 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.

7. The LNP of claim 1 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide;
(ii) a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide;
(iii) a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide; and
(iv) a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide.

8. The LNP of claim 7 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF.

9. The LNP of claim 7 , wherein the peptide linker is a Gly/Ser linker.

10. The LNP of claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 48.

11. The LNP of claim 1 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.

12. The LNP of claim 11 , wherein the 3′ UTR comprises a microRNA binding site, and wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site.

13. The LNP of claim 1 , wherein the mRNA comprises a chemically modified nucleotide.

14. The LNP of claim 13 , wherein the mRNA is fully modified with chemically modified uridines.

15. A LNP comprising a mRNA comprising an ORF comprising from 5′ to 3′:
(i) a nucleotide sequence encoding a signal peptide;
(ii) a nucleotide sequence encoding a human IL-12B polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(iii) a nucleotide sequence encoding a peptide linker;
(iv) a nucleotide sequence encoding a human IL-12A polypeptide comprising the amino acid sequence of SEQ ID NO: 3,
wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid.

16. The LNP of claim 15 , wherein the signal peptide is selected from: (i) a heterologous signal peptide; (ii) a human IL-12A signal peptide; and (iii) a human IL-12B signal peptide.

17. The LNP of claim 16 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.

18. The LNP of claim 15 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.

19. The LNP of claim 18 , wherein the 3′ UTR comprises a microRNA binding site, and wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site.

20. The LNP of claim 15 , wherein the mRNA comprises a chemically modified nucleotide.

21. The LNP of claim 20 , wherein the mRNA is fully modified with chemically modified uridines.

22. A LNP comprising a mRNA comprising an ORF comprising from 5′ to 3′:
(i) a nucleotide sequence encoding a signal peptide;
(ii) a nucleotide sequence encoding a human IL-12B polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(iii) a nucleotide sequence encoding a human IL-12A polypeptide comprising the amino acid sequence of SEQ ID NO: 3,
wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid.

23. The LNP of claim 22 , wherein the signal peptide is selected from: (i) a heterologous signal peptide; (ii) a human IL-12A signal peptide; and (iii) a human IL-12B signal peptide.

24. The LNP of claim 23 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.

25. The LNP of claim 22 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.

26. The LNP of claim 25 , wherein the 3′ UTR comprises a microRNA binding site, and wherein the microRNA binding site is a miR-122-3p or a miR-122-5p binding site.

27. The LNP of claim 22 , wherein the mRNA comprises a chemically modified nucleotide.

28. The LNP of claim 27 , wherein the mRNA is fully modified with chemically modified uridines.

29. The LNP of claim 1 , 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, and wherein the mRNA is fully modified with chemically modified uridines.

30. The LNP of claim 15 , 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, and wherein the mRNA is fully modified with chemically modified uridines.

31. The LNP of claim 22 , 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, and wherein the mRNA is fully modified with chemically modified uridines.

Assignees

• Modernatx Inc

Inventors

• Frederick Joshua
• Hewitt Susannah
• Bai Ailin
• Hoge Stephen
• Presnyak Vladimir
• Mcfadyen Iain
• Benenato Kerry
• Kumarasinghe Ellalahewage Sathyajith

Classifications

• A61K48/0008

  characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition · CPC title

• C07K14/5434

  IL-12 · CPC title

• A61K31/7115

  Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• A61K9/0019 Primary

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title