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1. WO2017060314 - 3' UTR SEQUENCES FOR STABILIZATION OF RNA

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Claims

1. A nucleic acid molecule comprising in the 5' → 3' direction of transcription:

(a) a promoter;

(b) a transcribable nucleic acid sequence or a nucleic acid sequence for introducing a transcribable nucleic acid sequence; and

(c) a nucleic acid sequence which, when transcribed under the control of the promoter (a), codes for a 3'-untranslated region in the transcript, said 3'-untranslated region comprising a nucleic acid sequence which is selected from the group consisting of:

(c-1) the nucleic acid sequence of the 3'- untranslated region of FCGRT, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-2) the nucleic acid sequence of the 3'- untranslated region of LSP1, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-3) the nucleic acid sequence of the 3'- untranslated region of CCL22, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-4) the nucleic acid sequence of the 3'- untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-5) the nucleic acid sequence of the 3'- untranslated region of PLD3, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-6) the nucleic acid sequence of the non- coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-7) the nucleic sequence of the 3'- untranslated region of HLA-DRB4, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

and

(c-8) any combination of two or more of the nucleic acid sequences, fragments and/or variants under (c-1), (c-2), (c-3), (c-4), (c-5), (c-6) and (c-7).

2. The nucleic acid molecule as claimed in claim 1, wherein the nucleic acid sequences (b) and (c) under the control of the promoter (a) can be transcribed to give a common transcript in which the nucleic acid sequence transcribed from the nucleic acid sequence (c) is active so as to increase the translation efficiency and/or the stability of the nucleic acid sequence transcribed from the transcribable nucleic acid sequence (b).

3. The nucleic acid molecule as claimed in claim 1 or 2, wherein (c-4) the nucleic acid sequence of the 3 '-untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence selected from SEQ ID NOs: 86 to 89, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

4. The nucleic acid molecule as claimed in any one of claims 1 to 3, wherein (c-4) the nucleic acid sequence of the 3'-untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 86, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

5. The nucleic acid molecule as claimed in any one of claims 1 to 4, wherein (c-6) the nucleic acid sequence of the non-coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence selected from SEQ ID NOs: 105 to 121, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

6. The nucleic acid molecule as claimed in any one of claims 1 to 5, wherein (c-6) the nucleic acid sequence of the non-coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 115, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

7. The nucleic acid molecule as claimed in any one of claims 1 to 6, wherein the nucleic acid sequence (c-8) comprises a combination of two or more identical or different nucleic acid sequences, fragments and/or variants under (c-1), (c-2), (c- 3), (c-4), (c-5), (c-6) and (c-7).

8. The nucleic acid molecule as claimed in any one of claims 1 to 7, wherein the nucleic acid sequence (c-8) comprises a combination of (c-4) the nucleic acid sequence of the 3'-untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment, and (c-6) the nucleic acid sequence of the non-coding RNA of MT- RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

9. The nucleic acid molecule as claimed in claim 8, wherein (c-4) the nucleic acid sequence of the 3'- untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment is located 5' to (c-6) the nucleic acid sequence of the non-coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

10. The nucleic acid molecule as claimed in claim 8 or 9, wherein the combination of (c-4) the nucleic acid sequence of the 3'-untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment, and (c-6) the nucleic acid sequence of the non-coding RNA of MT- RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 174, a fragment thereof, or a variant of said nucleic acid sequence or fragment.

11. The nucleic acid molecule as claimed in any one of claims 1 to 10, further comprising (d) a nucleic acid sequence which, when transcribed under the control of the promoter (a), codes for a nucleic acid sequence which is a polyadenyl sequence optionally comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides.

12. The nucleic acid molecule as claimed in claim 11, wherein said polyadenyl sequence comprises at least 20 A nucleotides, preferably at least 40, at least 80, at least 100 or at least 120 A nucleotides, preferably consecutive A nucleotides.

13. The nucleic acid molecule as claimed in claim 11 or 12, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides is a sequence, preferably an arbitrary sequence, of 2 or more consecutive nucleotides, wherein the first and the last nucleotide of said sequence of 2 or more consecutive nucleotides is a nucleotide other than an A nucleotide.

14. The nucleic acid molecule as claimed in any one of claims 11 to 13, wherein said nucleic acid sequence (d) is a nucleic acid sequence which, when transcribed under the control of the promoter (a), codes for a nucleic acid sequence which is a polyadenyl sequence comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides and which exhibits higher stability upon propagation of said nucleic acid molecule in Escherichia coli compared to a nucleic acid molecule which comprises instead of said nucleic acid sequence (d) a nucleic acid sequence (d)' which, when transcribed under the control of the promoter (a), codes for a polyadenyl sequence of the same length as said nucleic acid sequence which is a polyadenyl sequence comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides.

15. The nucleic acid molecule as claimed in any one of claims 11 to 14, wherein said nucleic acid sequence which is a polyadenyl sequence optionally comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotidescomprises at least 80 nucleotides, preferably at least 90 or 100 nucleotides.

16. The nucleic acid molecule as claimed in any one of claims 11 to 15, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides is located within a region from position 21 to position 80, preferably from position 21 to position 60, more preferably from position 31 to position 50 of said polyadenyl sequence.

17. The nucleic acid molecule as claimed in any one of claims 11 to 16, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides is preceeded by at least 20 A residues in said polyadenyl sequence and/or is followed by at least 20 A residues in said polyadenyl sequence.

18. The nucleic acid molecule as claimed in any one of claims 11 to 17, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides has a length of at least 3, at least 4, at least 5, at least 6, at least 8, preferably at least 10, more preferably at least 15 nucleotides.

19. The nucleic acid molecule as claimed in any one of claims 11 to 18, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides has a length of no more than 50, preferably no more than 30, more preferably no more than 20 nucleotides.

20. The nucleic acid molecule as claimed in any one of claims 11 to 19, wherein said sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides does not comprise more than 3, preferably no more than 2, preferably no consecutive A residues.

21. The nucleic acid molecule as claimed in any one of claims 11 to 20, wherein the nucleic acid sequences (b), (c) and (d) under the control of the promoter (a) can be transcribed to give a common transcript.

22. The nucleic acid molecule as claimed in claim 21, wherein the nucleic acid sequences transcribed from the nucleic acid sequences (c) and optionally (d) are active so as to increase the translation efficiency and/or the stability of the nucleic acid sequence transcribed from the transcribable nucleic acid sequence (b).

23. The nucleic acid molecule as claimed in any one of claims 11 to 22, wherein in the transcript said nucleic acid sequence which is a polyadenyl sequence optionally comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides is located at the 3' end.

24. The nucleic acid molecule as claimed in any one of claims 1 to 23, which is a closed circular molecule or a linear molecule.

25. The nucleic acid molecule as claimed in any one of claims 1 to 24, wherein the transcribable nucleic acid sequence comprises a nucleic acid sequence coding for a peptide or protein and the nucleic acid sequence for introducing a transcribable nucleic acid sequence is a multiple cloning site.

26. The nucleic acid molecule as claimed in any one of claims 1 to 25, further comprising one or more members selected from the group consisting of: (i) a reporter gene; (ii) a selectable marker; and (iii) an origin of replication.

27. The nucleic acid molecule as claimed in any one of claims 1 to 26, which is suitable, in particular after linearization, for in vitro transcription of RNA, in particular mRNA.

28. RNA which is obtainable by transcription, preferably in vitro transcription, using a nucleic acid molecule as claimed in any one of claims 1 to 27 as a template.

29. RNA comprising in the 5' → 3' direction:

(a) a 5'-untranslated region;

(b) a nucleic acid sequence coding for a peptide or protein; and

(c) a 3'-untranslated region, said 3'- untranslated region comprising a nucleic acid sequence which is selected from the group consisting of:

(c-1) the nucleic acid sequence of the 3'- untranslated region of FCGRT, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-2) the nucleic acid sequence of the 3'- untranslated region of LSP1, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-3) the nucleic acid sequence of the 3'- untranslated region of CCL22, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-4) the nucleic acid sequence of the 3'- untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-5) the nucleic acid sequence of the 3'- untranslated region of PLD3, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(c-6) the nucleic acid sequence of the non- coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment, (c-7) the nucleic sequence of the 3'- untranslated region of HLA-DRB4, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

and

(c-8) any combination of two or more of the nucleic acid sequences, fragments id/or variants under (c-1), (c-2), (c-3), (c-4), -5), (c-6) and (c-7).

30. The RNA as claimed in claim 29, further comprising (d) a nucleic acid sequence which is a polyadenyl sequence optionally comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides.

31. The RNA as claimed in claim 30, wherein said nucleic acid sequence (d) is located at the 3' end of said RNA.

32. The RNA as claimed in any one of claims 29 to 31, wherein the nucleic acid sequences (c) and optionally (d) are active so as to increase the translation efficiency and/or the stability of the nucleic acid sequence coding for a peptide or protein.

33. The RNA as claimed in any one of claims 29 to 32, further comprising (e) a 5' Cap.

34. A method of obtaining RNA, comprising:

(i) providing a nucleic acid molecule as claimed in any one of claims 1 to 27, and

(ii) transcribing RNA using the nucleic acid molecule as a template.

35. A method of obtaining a peptide or protein, comprising:

(i) obtaining RNA encoding the peptide or protein according to the method of claim 34, and

(ii) translating the RNA.

36. The method as claimed in claim 34 or 35, characterized in that it further comprises, prior to transcription of the nucleic acid molecule, cleavage of the nucleic acid molecule.

37. A method of obtaining RNA, comprising:

(i) coupling a nucleic acid sequence (b) which, when transcribed, codes for a 3'-untranslated region, at the 3' end of a transcribable nucleic acid sequence (a) comprising a nucleic acid sequence coding for a peptide or protein, and

(ii) transcribing the nucleic acid obtained, said 3'-untranslated region comprising a nucleic acid sequence which is selected from the group consisting of:

(b-1) the nucleic acid sequence of the 3'- untranslated region of FCGRT, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-2) the nucleic acid sequence of the 3'- untranslated region of LSP1, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-3) the nucleic acid sequence of the 3'- untranslated region of CCL22, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-4) the nucleic acid sequence of the 3'- untranslated region of AES, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-5) the nucleic acid sequence of the 3'- untranslated region of PLD3, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-6) the nucleic acid sequence of the non- coding RNA of MT-RNR1, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

(b-7) the nucleic sequence of the 3'- untranslated region of HLA-DRB4, a fragment thereof, or a variant of said nucleic acid sequence or fragment,

and

(b-8) any combination of two or more of the nucleic acid sequences, fragments and/or variants under (b-1), (b-2), (b-3), (b-4), (b-5), (b-6) and (b-7).

38. The method as claimed in claim 37, wherein the nucleic acid sequences (a) and (b) can be transcribed to give a common transcript in which the nucleic acid sequence transcribed from the nucleic acid sequence (b) is active so as to increase the translation efficiency and/or the stability of the nucleic acid sequence transcribed from the transcribable nucleic acid sequence (a).

39. The method as claimed in claim 37 or 38, which further comprises coupling a nucleic acid sequence (c) which, when transcribed, codes for a nucleic acid sequence which is a polyadenyl sequence optionally comprising within the polyadenyl sequence a sequence of one or more consecutive nucleotides containing nucleotides other than A nucleotides, at the 3' end of the nucleic acid sequence (b).

40. The method as claimed in claim 39, wherein the nucleic acid sequences (a), (b), and (c) can be transcribed to give a common transcript in which the nucleic acid sequences transcribed from the nucleic acid sequences (b) and, optionally, (c) are active so as to increase the translation efficiency and/or the stability of the nucleic acid sequence transcribed from the transcribable nucleic acid sequence (a).

41. A method of obtaining a peptide or protein, comprising:

(i) obtaining RNA by the method of any one of claims 37 to 40, and

(ii) translating the RNA.

42. The method as claimed in any one of claims 34 to 41, wherein transcription is carried out in vitro.

43. RNA obtainable by the method as claimed in any one of claims 34, 36 to 40 and 42.

44. A method of obtaining a peptide or protein, comprising translating the RNA as claimed in any one of claims 28 to 33 and 43.

45. Use of the RNA as claimed in any one of claims 28 to 33 and 43 for transfecting a host cell.

46. The use as claimed in claim 45, wherein the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

47. Use of the RNA as claimed in any one of claims 28 to 33 and 43 for vaccination.

48. Use of the RNA as claimed in any one of claims 28 to 33 and 43 for reprogramming somatic cells to cells having stem cell characteristics.