Method for designing RIG-I ligands

Abstract

The present invention belongs to the field of biotechnology and pharmaceuticals. The present inventors found a sequence motif for identifying potent RIG-I agonists. Accordingly, the present invention is directed to a method for producing RIG-I agonists, the RIG-I agonists produced by said methods, and uses of said RIG-I agonists, as defined in the claims.

Claims

1. A method for producing a RIG-I agonist, comprising the steps of (a) preparing a first polyribonucleotide with 21-300 nucleotides in length, which polyribonucleotide starts at the 5′ end with a sequence selected from TABLE-US-00023 (SEQ ID NO: 12) 5′-gbucndnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 13) 5′-gucuadnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 14) 5′-guagudnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 15) 5′-gguaadnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 16) 5′-ggcagdnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 17) 5′-gcuucdnwnnnnnnnnwnsnn-3′, (SEQ ID NO: 18) 5′-gcccadnwnnnnnnnnwnsnn-3′, and (SEQ ID NO: 19) 5′-gcgcudnwnnnnnnnnwnsnn-3′; (b) preparing a second polyribonucleotide with 21-300 nucleotides in length which is at least 80% complementary to the first polyribonucleotide of step (a) over the whole length of the first polyribonucleotide of step (a), and which, when annealed to the first polyribonucleotide of step (a), exhibits either no 3′ overhang, or a 3′ overhang of 1 or 2 nucleotides; and (c) annealing the first polyribonucleotide of step (a) with the second polyribonucleotide of step (b), thereby obtaining a RIG-I agonist; wherein “b” denotes g, c, or u, “d” denotes a, g, or u, “w” denotes a, or u, “s” denotes g or c, and “n” denotes any nucleotide base.

2. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 6 is u.

3. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 7 is g.

4. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 6 is g, and the ribonucleotide at position 7 is c.

5. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 8 is a.

6. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 9 is a.

7. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 17 is u, and wherein the sequence at the 5′end of the polyribonucleotide in step (a) is selected from TABLE-US-00024 (SEQ ID NO: 95) 5′-gbucndnwnnnnnnnnunsnn-3′, (SEQ ID NO: 96) 5′-gucuadnwnnnnnnnnunsnn-3′, (SEQ ID NO: 97) 5′-guagudnwnnnnnnnnunsnn-3′, (SEQ ID NO: 98) 5′-gguaadnwnnnnnnnnunsnn-3′, (SEQ ID NO: 99) 5′-ggcagdnwnnnnnnnnunsnn-3′, (SEQ ID NO: 100) 5′-gcuucdnwnnnnnnnnunsnn-3′, (SEQ ID NO: 101) 5′-gcccadnwnnnnnnnnunsnn-3′, and (SEQ ID NO: 102) 5′-gcgcudnwnnnnnnnnunsnn-3′.

8. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 18 is u.

9. The method of claim 1, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 19 is c.

10. The method of claim 1, wherein the sequence at the 5′ end of the polyribonucleotide in step (a) is selected from TABLE-US-00025 (SEQ ID NO: 20) 5′-gbucnugaannnnnnnuucnn-3′, (SEQ ID NO: 21) 5′-gucuaugaannnnnnnuucnn-3′, (SEQ ID NO: 22) 5′-guaguugaannnnnnnuucnn-3′, (SEQ ID NO: 23) 5′-gguaaugaannnnnnnuucnn-3′, (SEQ ID NO: 24) 5′-ggcagugaannnnnnnuucnn-3′, (SEQ ID NO: 25) 5′-gcuucugaannnnnnnuucnn-3′, (SEQ ID NO: 26) 5′-gcccaugaannnnnnnuucnn-3′, (SEQ ID NO: 27) 5′-gcgcuugaannnnnnnuucnn-3′, and (SEQ ID NO: 66) 5′-gbucnugaaannnnnuuucnn-3′.

11. The method of claim 1, wherein the polyribonucleotide in step (a) has a length of at most 30 nucleotides, or wherein the complementary polyribonucleotide in step (b) is at least 90% complementary to the first polyribonucleotide of step (a) over the whole length of the first polyribonucleotide of step (a), or both the polyribonucleotide in step (a) has a length of at most 30 nucleotides and the complementary polyribonucleotide in step (b) is at least 90% complementary to the first polyribonucleotide of step (a) over the whole length of the first polyribonucleotide of step (a).

12. The method of claim 1, wherein the annealed polyribonucleotide of step (c) has two blunt ends, and a length of 24 nucleotides.

13. The method of claim 12, wherein in the polyribonucleotide of step (a) the ribonucleotide sequence at positions 20-24 is selected from 5′-ngavc-3′, 5′-uagac-3′, 5′-acuac-3′, 5′-uuacc-3′, 5′-cugcc-3′, 5′-gaagc-3′, 5′-ugggc-3′ and 5′-agcgc-3′, wherein “v” denotes a, g or c.

14. The method of claim 12, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 6 is g, the ribonucleotide at position 7 is a, and the ribonucleotide at position 8 is a.

15. The method of claim 14, wherein the ribonucleotide at position 9 is a.

16. The method of claim 12, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 16 is u.

17. The method of claim 12, wherein in the sequence of the polyribonucleotide of step (a) the ribonucleotide at position 17 is u the ribonucleotide at position 18 is g, and the ribonucleotide at position 19 is c.

18. The method of claim 12, wherein in the sequence of the polyribonucleotide of step (a) the sequence at position 6-24 is selected from TABLE-US-00026 (SEQ ID NO: 3) 5′-ugaannnnnnnuucngavc-3′, (SEQ ID NO: 4) 5′-ugaannnnnnnuucngavc-3′, (SEQ ID NO: 5) 5′-ugaannnnnnuuucngavc-3′, (SEQ ID NO: 6) 5′-gaaannnnnnnuucngavc-3′, (SEQ ID NO: 67) 5′-gaaannnnnnnuucngavc-3′, and (SEQ ID NO: 68) 5′-gaaannnnnnuuucngavc-3′; or wherein the first RNA sequence of step (a) is selected from (SEQ ID NO: 7) 5′-gbucnugaannnnnnnuucnnnnn-3′, (SEQ ID NO: 8) 5′-gbucnugaannnnnnnuucngavc-3′, (SEQ ID NO: 9) 5′-gbucnugaannnnnnnuucngavc-3′, (SEQ ID NO: 10) 5′-gbucnugaannnnnnuuucngavc-3′, (SEQ ID NO: 11) 5′-gbucngaaannnnnnnuucngavc-3′, (SEQ ID NO: 69) 5′-gbucngaaannnnnnnuucngavc-3′ and (SEQ ID NO: 70) 5′-gbucngaaannnnnnuuucngavc-3′. wherein “v” denotes a, g or c.

19. The method of claim 1, wherein the polyribonucleotide prepared in step (a) has a mono-, di-, or triphosphate or respective analogue attached to its 5′ end.

20. The method of claim 19, wherein the polyribonucleotide prepared in step (a) has a triphosphate attached to its 5′ end.

21. The method of claim 1, wherein the complementary polyribonucleotide prepared in step (b) has a mono-, di-, or triphosphate or respective analogue attached to its 5′ end.

22. The method of claim 21, wherein the complementary polyribonucleotide prepared in step (b) has a triphosphate attached to its 5′ end.

23. The method of claim 1, wherein the first RNA sequence of step (a) is TABLE-US-00027 (SEQ ID NO: 104) 5′-guucugcaaucagcuauacguuau-3′ or (SEQ ID NO: 103) 5′-guucugcaaucagcuaaacguuau-3′.

24. The method of claim 23, wherein the first RNA sequence of step (a) is TABLE-US-00028 (SEQ ID NO: 108) 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ or (SEQ ID NO: 107) 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′, the nucleotide indexed m is 2′-O-methylated; the nucleotide indexed f is 2′-fluoro; the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond; and 3P-5′ is a 5′-triphosphate.

25. The method of claim 23, wherein the second RNA sequence of step (b) is fully complementary to the first RNA sequence of step (a) and is TABLE-US-00029 (SEQ ID NO: 105) 5′-auaacguuuagcugauugcagaac-3′; or (SEQ ID NO: 106) 5′-aaauaacguuuagcugauugcagaac-3′; or (SEQ ID NO: 115) 5′-auaacguauagcugauugcagaac-3′; or (SEQ ID NO: 116) 5′-aaauaacguauagcugauugcagaac-3′.

26. The method of claim 25, wherein the second RNA sequence of step (b) is TABLE-US-00030 (SEQ ID NO: 109) 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 110) 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 111) 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 112) 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 117) 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 118) 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 119) 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 120) 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′; wherein the nucleotide indexed m is 2′-O-methylated; the nucleotide indexed f is 2′-fluoro; the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond; and 3′-P is a 3′-monophosphate.

27. The method of claim 1, wherein (I) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence of step (b) is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 117); or (II) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence of step (b) is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 118); or (III) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence of step (b) is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 119); or (IV) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence of step (b) is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 120); or (V) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence of step (b) is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 109); or (VI) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence of step (b) is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 110); or (VII) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence of step (b) is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgcuu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 111); or (VIII) the first RNA sequence of step (a) is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence of step (b) is 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 112).

28. A RIG-I agonist, obtainable by the method of claim 1; wherein the first RNA sequence is TABLE-US-00031 (SEQ ID NO: 104) 5′-guucugcaaucagcuauacguuau-3′ or (SEQ ID NO: 103) 5′-guucugcaaucagcuaaacguuau-3′.

29. The RIG-I agonist of claim 28, wherein the RIG-I agonist is a polyribonucleotide which comprises at least one synthetic or modified internucleoside linkage, selected from phosphodiester, phosphorothioate, N3 phosphoramidate, boranophosphate, 2,5-phosphodiester, amide-linked, phosphonoacetate (PACE), morpholino, peptide nucleic acid (PNA), or a mixture thereof, provided the linkage(s) do not compromise the type I IFN-inducing activity of the polyribonucleotide.

30. The RIG-I agonist of claim 29, wherein the polyribonucleotide comprises phosphorothioate linkage(s), pyrophosphate linkage(s), or both.

31. The RIG-I agonist of claim 28, wherein the RIG-I agonist is a polyribonucleotide which comprises at least one modified nucleotide selected from pseudouridine, 2-thiouridine, 2′-fluorine-dNTP, 2′-O-methylated NTP.

32. The RIG-I agonist of claim 28, wherein the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) or 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107), wherein the nucleotide indexed m is 2′-O-methylated; the nucleotide indexed f is 2′-fluoro; the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond; and 3P-5′ is a 5′-triphosphate.

33. The RIG-I agonist of claim 28, wherein the second RNA sequence is fully complementary to the first RNA sequence and is TABLE-US-00032 (SEQ ID NO: 105) 5′-auaacguuuagcugauugcagaac-3′; or (SEQ ID NO: 106) 5′-aaauaacguuuagcugauugcagaac-3′; or (SEQ ID NO: 115) 5′-auaacguauagcugauugcagaac-3′; or (SEQ ID NO: 116) 5′-aaauaacguauagcugauugcagaac-3′.

34. The RIG-I agonist of claim 33, wherein the second RNA sequence is TABLE-US-00033 (SEQ ID NO: 109) 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 110) 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 111) 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 112) 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 117) 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 118) 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′, or (SEQ ID NO: 119) 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P, or (SEQ ID NO: 120) 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′; wherein the nucleotide indexed m is 2′-O-methylated; the nucleotide indexed f is 2′-fluoro; the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond; and 3′-P is a 3′-monophosphate.

35. The RIG-I agonist of claim 33, wherein (I) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 117); or (II) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 118); or (III) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 119); or (IV) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 120); or (V) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 109); or (VI) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 110); or (VII) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 111); or (VIII) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 112).

36. A RIG-I agonist, wherein (I) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 117); or (II) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 118); or (III) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 119); or (IV) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 108) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macgua.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 120); or (V) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 109); or (VI) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 110); or (VII) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′-P (SEQ ID NO: 111); or (VIII) the first RNA sequence is 3P-5′-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu-3′ (SEQ ID NO: 107) and the second RNA sequence is 5′-aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-3′ (SEQ ID NO: 112).

37. A new pharmaceutical composition comprising at least one RIG-I agonist of claim 28, and a pharmaceutically acceptable carrier.

38. The pharmaceutical composition of claim 37, further comprising at least one agent selected from an anti-tumor agent, an immunostimulatory agent, an anti-viral agent, an anti-bacterial agent, a checkpoint-inhibitor, retinoic acid, IFN-α, and IFN-β.

39. The pharmaceutical composition of claim 37, wherein said composition is a vaccine composition.

40. A vaccine adjuvant, comprising a pharmaceutical composition according to claim 37.

41. An ex vivo method for inducing type I IFN production in a cell, comprising the step of contacting a cell expressing RIG-I with at least one RIG-I agonist according to claim 28.

42. A pharmaceutical composition comprising at least one RIG-I agonist of claim 36, and a pharmaceutically acceptable carrier.

43. An ex vivo method for inducing type I IFN production in a cell, comprising the step of contacting a cell expressing RIG-I with at least one RIG-I agonist according to claim 36.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Screening selected 5-mer sequences fused a 19-mer tail sequence (UGGAUGGUUGGCUAGGAUA) for IFNα induction in PBMCs.

(2) FIG. 2: Confirmation of suitability of 5-mer design in 2 independent 19-mer tail sequences (random: UGGAUGGUUGGCUAGGAUA (SEQ ID NO: 28); 121212 tail: UGACCCUGAAGUUCAUCUU (SEQ ID NO: 29)).

(3) FIG. 3: Permutation of the 121212 tail (UGACCCUGAAGUUCAUCUU (SEQ ID NO: 29)) by introducing 4-mer sequences from a random tail (UGGAUGGUUGGCUAGGAUA (SEQ ID NO: 28)) as indicated in (A) and screening for IFNα induction (B).

(4) FIG. 4: Permutation of single nucleotides within the 121212 tail sequence (UGACCCUGAAGUUCAUCUU (SEQ ID NO: 29)) and screening for IFNα induction (A) and comprehensive analysis of the role of position 6-8 and positions 17-19 for IFNα induction (B).

(5) FIG. 5: Analyzing the role of a G/C or C/G doublet at position 6/7.

(6) FIG. 6: Schematic overview of structural features required for optimal RIG-I agonism and detrimental sequence pattern.

(7) FIG. 7: The polyribonucleotides of Example 4 were tested for RIG-I selectivity. While the tested polyribonucleotides were capable of inducing IFNα (upper panel), no IFNα induction was obtained via TLR7 (middle panel) demonstrating a high selectivity of the tested ribonucleotides for RIG-I. Ribonucleotide 122212 was used as a positive control. The single strands of SEQ ID NO 107-112 and 117-120 did not activate TLR8 (lower panel). Ribonucleotide 122212 has the same sequence as ribonucleotide 121212, but carries a 5′-triphosphate in the sense strand.

EXAMPLES

(8) Material and Methods

(9) Cell Culture

(10) The human airway epithelial cell line A549 and the human HPV16.sup.+ cervix epithelial cell line CaSki were both obtained from Cell Lines Services (Germany). A549-PGK-EGFP cells were generated at Cellomics Technology, LLC (Halethorpe, Md., USA). Human primary peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats obtained from healthy volunteers according to standard protocols (Schuberth-Wagner et al., 2015, Immunity). PBMCs (2.6×10.sup.6 cells/ml) were seeded in 96-well plates and maintained in RPMI1640 supplemented with 10% FCS, 1.5 mM L-glutamine and 1× penicillin/streptomycin. In some experiments, PBMCs were pre-treated with 2.5 mg/ml chloroquine (Sigma Aldrich) for at least 1 hr to prevent endosomal TLR activation. All cell culture reagents were obtained from Gibco.

(11) Cell Stimulation

(12) Chemically synthesized RNA oligonucleotides were purchased from Biomers (Ulm, Germany) and Axolabs (Kulmbach, Germany). RNA was transfected into cells using Lipofectamine 2000 according to manufacturers instructions (Invitrogen). PBMCs were stimulated once and conditioned medium was collected after 17 hrs. CaSki cells were stimulated once or twice depending on the duration of the experiment (3 or 6d). For the long term experiment (6d), CaSki cells were treated on d0 and d3.

(13) Flowcytometric Quantitation of Intracellular EGFP

(14) FACS-based quantitation of intracellular EGFP levels was performed according to standard protocols. Briefly, after stimulation for the indicated time points, A549-PGK-EGFP cells were collected, resuspended in PBS and subjected to flowcytometric analysis. Changes in fluorescence signals were measured using an Attune NxT acoustic focusing cytometer (Life Technologies).

(15) XTT Assay To monitor changes of the cellular metabolic activity, the XTT assay was used according to the manufacturers instructions (Roche).

(16) Real-Time RT-PCR

(17) Steady state mRNA levels of HPV16E7 and IFNβ were quantified by real-time fluorescence detection using SYBR Select Master Mix (Life Technologies). Reactions in duplicate were analyzed in a Quant Studio 6 flex (Life Technologies). Specific primer pairs were as follows: HPV16E7 forward, 5′-AGTGTGACTCTACGCTTCGG-3 (SEQ ID NO: 30) and reverse, 5′-TGTGCCCATTAACAGGTCTT-3 (SEQ ID NO: 31), hIFNβ forward 5′-GTCACTGTGCCTGGACCATA-3′ (SEQ ID NO: 32) and reverse, 5′-AGAGGCACAGGCTAGGAGAT-3 (SEQ ID NO: 33), and hβ-Actin forward 5′-GAGACCGCGTCCGCC-3 (SEQ ID NO: 34) and reverse, 5′-ATCATCCATGGTGAGCTGGC-3 (SEQ ID NO: 35).

(18) IFNα ELISA

(19) Conditioned cell culture supernatant derived from activated PBMCs was harvested at 17 hrs time point. Quantitation of IFNα levels in cell culture supernatant was performed using the human IFN alpha matched antibody pairs ELISA (eBioscience, San Diego, Calif., USA).

Example 1-5′-Sequences Influence on Immune Activation

(20) Nucleic acid sensors efficiently trigger anti-viral and anti-cancer immune pathways to strengthen the body's defense mechanisms. Pharmacological activation of nucleic acid sensors such as TLRs and RIG-I emerged them as attractive targets to recover host homeostasis (Junt and Barchet, 2015, Nat Rev Immunol). Recent studies highlight structural features determining RIG-I activation: the phosphorylation status at the very 5′ end (Schlee et al, Goubau et al), the oligonucleotide length (Schlee) and the 5′ nucleotide (Schlee).

(21) Structural determinants distinguishing strong from weak RIG-I ligands remain largely elusive and thus we aimed to analyze the influence of different 5-mer sequences at the very 5′-end sensed by RIG-I's basic patch region. Therefore, sixty 5-mer sequences with a G/C content ranging from 40-80% were generated randomly Subsequently, all 5-mer sequences were linked to a 19-mer random tail (5′-UGGAUGGUUGGCUAGGAUA-3′ (SEQ ID NO: 28)) constituting the 24-mer sense strand. The complementary anti-sense strand had a 5′ two nucleotide overhang to allow directed recognition of the random 5-mer sequences through RIG-I. Screening revealed that only 12 out of 60 RIG-I ligands induced good IFNα release (threshold 50 pg/ml) from primary human PBMCs (FIG. 1, dark grey bars). Moreover, the effect appeared to be independent of the G/C content (FIG. 1). Comparative analysis of all 12 good 5-mer sequences showed that certain nucleotides at particular positions were preferred and a consensus motif for the 5-mer sequence (5′-G.sub.1-noA.sub.2-U.sub.3-C.sub.4-N.sub.5-3′) was elaborated:

(22) TABLE-US-00015 5-mer sequences G G U C C G U U C U G U C U A G U A G U G G U A A G C U C C G G C A G G C U C U G C U U C G C C C A G U U C C G C G C U Consensus G no A U C N

(23) Of note, 41% of all 12 good 5-mer sequences were completely covered by the 5-mer consensus motif, whereas none of the weak 5-mer sequences contained this motif (FIG. 1).

(24) To further substantiate our findings we linked both strong and weak 5-mer sequences to a set of independent 19-mer tail sequences (5′-UGACCCUGAAGUUCAUCUU-3′ (SEQ ID NO: 36), 5′-UGACCCUGAAGUUCAUCU-3′ (SEQ ID NO: 37), 5′-UCAAGGUGAACUUCAAGAU-3′ (SEQ ID NO: 38), 5′-GGCUACGUCCAGGAGCGCA-3′ (SEQ ID NO: 39)) and analyzed IFNα release from PBMCs. Indeed, we validated our previous findings and showed that 5-mer sequences considered as good were superior to those considered as weak in different 19-mer tail settings (FIGS. 2, 6B/C and 7B). Together, our data clearly demonstrated optimization of the 5′ 5-mer sequence can promote RIG-I-mediated immune activation.

Example 2—Functional Boxes Reside within the Tail Sequence and Regulate Immune Activation

(25) The role of tail sequence inherent features had not been investigated as of yet. Thus, to explore whether functional structural elements within 19-mer tail sequence exist and how these might shape the immune response, the 121212 tail sequence was modified stepwise. Single or more 4-mer sequences were substituted by corresponding 4-mer cassettes derived from the random tail (5′-UGGAUGGUUGGCUAGGAUA-3′ (SEQ ID NO: 28)) (FIG. 3A). Analysis of these novel dsRNA sequences revealed that 4-mer substitutions at positions 8-11, 16-19, 20-23, 8-15, 16-23, 8-11/16-19, 8-11/20-23 and 12-19 (counted from the very 5′ end of the RNA included the 5-mer sequence) are detrimental and negatively regulate RIG-I-induced IFNα release (FIG. 3B). We then set out to narrow down crucial sequence sections by changing single nucleotides within the 19-mer 121212 tail. In particular, nucleotide substitutions at the positions 6, 8, 17 and 19 within the 5′-GACGC UGACCCUGAAGUUCAUCUU-3′ (SEQ ID NO: 40) led to reduced IFNα induction (FIGS. 4A and B). Moreover, substitution of “u” at position 6 of 121212 for a “c” (final RNA sequence: 5′-GACGC CGACCCUGAAGUUCAUCUU-3′ (SEQ ID NO: 71)) reduced IFNα induction by app. 50% as compared to the parent 121212 (FIG. 4B), indicating detrimental effects of c.sub.6/g.sub.7 doublets. This is further supported by data shown in FIG. 5, as a set of different dsRNAs having a c/g doublet at position 6/7 showed no up-regulation of IFNα. However, g6/c7 doublets do not compromise the IFNα induction (FIG. 5).

(26) An adenosine at position 9 elevates the IFNα response (FIG. 4A). Together, sequence specific properties determine the agonist's inherent potential to promote inflammation.

Example 3—Combining Structural Features to Create a Design Rule for Optimal RIG-I Agonism

(27) Intense studies on optimal 5′ 5-mer sequences and intramolecular nucleotide substitutions revealed important structural elements accounting for RIG-I agonism as described above. All elements identified were applied to a 24-mer sequence as shown in FIG. 6. The very 5′ consensus 5-mer sequence is shown in the very left box. Regions 6-8 and 17-19 were assigned as box 1 (middle) and box 2 (very right), respectively and accepted nucleotide substituents with minor effects on immune stimulation are shown below. Moreover, detrimental nucleotide doublets in box 1 and their exact positions are indicated. Furthermore, adenosine at position 9 is highlighted (FIG. 6).

(28) Here we presented the development of a novel design rule to predict highly versatile RIG-I-ligands. We identified a consensus 5-mer cassette (5′-G.sub.1-noA.sub.2-U.sub.3-C.sub.4-N.sub.5-3′) and a 19-mer tail sequence comprising two 3-nt boxes located at positions 6-8 (box 1) and 17-19 (box 2) within a 24-mer oligonucleotide backbone counted from the 5′ end of the sense strand. Moreover, we revealed nucleotide doublets within box 1 that are detrimental and strongly counteract RIG-I-induced immunity.

Example 4—Assessment of Polyribonucleotides Obtained by the Design Rule of the Present Disclosure

(29) The following polyribonucleotides were designed following the design rule for RIG-I agonists of the present disclosure:

(30) TABLE-US-00016 Oligo- nucleo- tides Sequence (5′-3′) SEQ ID NO 1 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu 107 Antisense a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-P 109 2 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu 107 Antisense a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac 110 3 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu 107 Antisense aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-P 111 4 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuaaacguu.sub.PTOa.sub.PTOu 107 Antisense aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac 112 5 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu 108 Antisense a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-P 109 6 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu 108 Antisense a.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac 110 7 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu 108 Antisense aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac-P 111 8 Sense 3P-g.sub.PTOu.sub.PTOucu.sub.mgcaa.sub.fucag.sub.fcuauacguu.sub.PTOa.sub.PTOu 108 Antisense aaa.sub.PTOu.sub.PTOa.sub.macguu.sub.fuagc.sub.fugauugcagaac 112
the nucleotide indexed m is 2′-O-methylated;
the nucleotide indexed f is 2′-fluoro;
the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond;
—P is a 3′-monophosphate;
3P— is a 5′triphosphate; and
—OH is a 3′-OH.

(31) In certain assays, the following prior art RIG-I agonist (WO 2014/049079 A1) was used for comparison

(32) TABLE-US-00017 Sequence (5′-3) SEQ ID NO 3P-GFP2 1S2F Sense 3P-gacgcug.sub.facccugaa.sub.mguucauc.sub.PTO(u.sub.f).sub.PTOu 113 Antisense a.sub.PTOa.sub.PTOgaugaacuuc.sub.fagggucagcg.sub.muc 114
the nucleotide indexed m is 2′-O-methylated;
the nucleotide indexed f is 2′-fluoro;
the index PTO between two nucleotides indicates that said two nucleotides are linked by a phosphothioate bond; and
3P— is a 5′triphosphate.
Metabolic Activity in Human Tumor Cells

(33) The human adenocarcinoma cell line HT29 was obtained from Cell Lines Services (Germany), and the human melanoma cell line D04mel is available through the Australasian Biospecimen Network (Oncology) Cell Line Bank at the QIMR Berghofer Medical Research Institute. Human tumor cell lines D04 and HT29 were seeded at a density of 1×10.sup.4 cells per well of a 96-well plate. Chemically synthesized RNA oligonucleotides were purchased from Biomers (Ulm, Germany), Axolabs (Kulmbach, Germany) or were produced internally. RNA was transfected into the cells using Lipofectamine 2000 according to manufacturer's instructions (Invitrogen). To monitor changes of the cellular metabolic activity, the cell cultures were assayed after 68 hours for metabolic activity using the XTT assay according to the manufacturer's instructions (Roche). The results are shown in the following table:

(34) TABLE-US-00018 Metabolic activity IC.sub.50 in D04 cells in HT29 cells Oligonucleotides [×10.sup.−4 nM] [×10.sup.−2 nM] 1 0.3 4 2 0.9 3 3 1 2 4 0.03 0.9 5 0.4 2 6 0.07 0.3 7 0.3 0.6 8 0.6 1 3P-GFP2 1S2F 1

(35) The IC.sub.50 of oligonucleotides-5, 6, 7 and 8 is generally lower as compared to oligonucleotides 1, 2, 3 and 4. In D04 cells, the polyribonucleotides designed in accordance with the design rule of the present disclosure show an IC.sub.50 which is lower than the IC.sub.50 of the prior art RIG-I agonist 3P-GFP2 1S2F.

(36) Metabolic Activity in Murine Tumor Cells

(37) Murine cell lines CT26 and B16 were seeded at a density of 4×10.sup.4 cells per well of a 96-well plate. Chemically synthesized RNA oligonucleotides were purchased from Biomers (Ulm, Germany), Axolabs (Kulmbach, Germany) or were produced internally. RNA was transfected into the cells using Lipofectamine 2000 according to manufacturer's instructions (Invitrogen). To monitor changes of the cellular metabolic activity, the cell cultures were assayed after 68 hours for metabolic activity using the XTT assay according to the manufacturer's instructions (Roche). The results are shown in the following table:

(38) TABLE-US-00019 Metabolic activity IC.sub.50 in B16 cells in HT29 cells Oligonucleotides [×10.sup.−2 nM] [×10.sup.−2 nM] 1 0.05 0.4 2 0.06 0.3 3 0.04 0.4 4 0.04 0.2 5 0.004 0.4 6 0.0008 0.2 7 0.007 0.3 8 0.0001 0.001 3P-GFP2 1S2F 7 4

(39) The IC.sub.50 of oligonucleotides 5, 6, 7 and 8 is lower as compared to oligonucleotides 1, 2, 3 and 4. The polyribonucleotides designed in accordance with the design rule of the present disclosure show an IC.sub.50 which is lower than the IC.sub.50 of the prior art RIG-I agonist 3P-GFP2 1S2F.

(40) Induction of Apoptosis in Human D04 Cells

(41) To monitor apoptosis, human D04 cells were seeded at a density of 3×10.sup.4 cells/well into 96 well plates and were treated with agonists as described above. After 48 hours both adherent and floating cells were collected. Cells were washed with cell staining buffer and finally resuspended in Annexin V binding buffer. 2.5 μl/well APC-Annexin V and 2.5 μl/well 7-AAD were added. After 15 min incubation, 100 μl/well Annexin V binding buffer was added and the cells were analyzed using an Attune NxT acoustic focusing cytometer (Life Technologies). The results are shown in the following table:

(42) TABLE-US-00020 Induction of Apoptosis EC.sub.50 AV+/AAD− AV+ AAD+ AV− AAD− Oligonucleotides [nM] [nM] [nM] 1 0.04 0.1 0.05 4 0.03 0.3 0.05 5 0.01 0.01 0.01 8 0.1 0.04 0.01 3P-GFP2 1S2F 0.06 0.2 0.05
Induction of Apoptosis in Murine Colon 26 Cells

(43) To monitor apoptosis, murine CT26 cells were seeded at a density of 3×10.sup.4 cells/well into 96 well plates and were treated with agonists as described above. After 48 hours both adherent and floating cells were collected. Cells were washed with cell staining buffer and finally resuspended in Annexin V binding buffer. 2.5 μl/well APC-Annexin V and 2.5 μl/well 7-AAD were added. After 15 min incubation, 100 μl/well Annexin V binding buffer was added and the cells were analyzed using an Attune NxT acoustic focusing cytometer (Life Technologies). The results are shown in the following table:

(44) TABLE-US-00021 Induction of Apoptosis EC.sub.50 AV+/AAD+ AV− AAD− Oligonucleotides [nM] [nM] 1 0.1 0.2 4 0.06 0.07 5 0.2 0.1 8 0.2 0.1 3P-GFP2 1S2F 0.2 0.2
EC.sub.50 in Human PBMC (IFNα ELISA)

(45) Human primary peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats obtained from healthy volunteers according to standard protocols (Schuberth-Wagner et al., 2015, Immunity). PBMCs (4×10.sup.5 cells/well) were seeded in 96-well plates and maintained in RPMI1640 supplemented with 10% FCS, 1.5 mM L-glutamine and 1× penicillin/streptomycin. All cell culture reagents were obtained from Gibco. Chemically synthesized RNA oligonucleotides were purchased from Biomers (Ulm, Germany), Axolabs (Kulmbach, Germany) or were produced internally. RNA was transfected into cells using Lipofectamine 2000 according to manufacturers instructions (Invitrogen). PBMCs were stimulated once and conditioned medium was collected after 17 hrs. Quantitation of IFNα levels in cell culture supernatant was performed using the human IFN alpha matched antibody pairs ELISA (eBioscience, San Diego, Calif., USA). The results are shown in the following table:

(46) TABLE-US-00022 EC.sub.50 in PBMC IFNα ELISA Oligonucleotides [nM] 1 0.09 2 0.09 3 0.07 4 0.08 5 0.03 6 0.06 7 0.03 8 0.1 3P-GFP2 1S2F 0.07
5, 6 and 7 show a EC.sub.50 which is lower than for 1, 2 and 3, and which is lower than for 3P-GFP2 1S2F.

(47) In order to assay the polyribonucleotides for their RIG-I selectivity, RNA was transfected into PBMCs using poly-L-arginine (Sigma Aldrich). RNA and poly-L-arginine were allowed to complex for 0 min and 20 min to trigger TLR7 and TLR8 activation, respectively. Supernatant was pulled after 17 hrs and the cytokine of interest was analyzed. Quantitation of IFNα levels in cell culture supernatant was performed using the human IFN alpha matched antibody pairs ELISA (eBioscience, San Diego, Calif., USA). As an indirect measure of TLR8 activation, IL-12p70 was quantified using the BD kit #555183. As can be seen in FIG. 7, induction of IFNα was specifically triggered via RIG-I but not by TLR 7 or TLR8. Ribonucleotide 122212 was used as a positive control.

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