Nucleic acid molecules

Abstract

The present disclosure relates to a nucleic acid molecule comprising 5'-UTR and / or 3'-UTR sequences that yield high translation levels. Aspects of the disclosure further relate to nucleic acid molecules suitable for use as a vaccine in the treatment and prevention of infectious diseases, including those caused by a coronavirus, compositions comprising said nucleic acid molecules and methods of treating or preventing infectious diseases.

Description

This disclosure relates to a nucleic acid molecule comprising a 5'-UTR and / or 3'-UTR sequence that produces a high level of translation. Aspects of this disclosure further relate to nucleic acid molecules suitable for use as vaccines for the treatment and prevention of infectious diseases, including those caused by coronaviruses, compositions comprising said nucleic acid molecules, and methods for treating or preventing infectious diseases. Abstract

Claims

Claims1. A nucleic acid molecule comprising a 5’ untranslated region (5’-UTR), a coding sequence and a 3’ untranslated region (3’-UTR), wherein the coding sequence is operably linked to the 5’-UTR and the 3’-UTR and wherein(i) the 5’-UTR comprises a sequence derived from a 5’-UTR of human chitinase-1 (CHIT1) and the 3’-UTR comprises a sequence derived from a 3’-UTR of human citrate synthase (CS);(ii) the 5’-UTR comprises a sequence derived from a 5’-UTR of human protein kinase cAM P-activated catalytic subunit beta (PRKACB) and the 3’-UTR comprises a sequence derived from a 3’-UTR of human chitinase-1 (CHIT1);(iii) the 5’-UTR comprises a sequence derived from a 5’-UTR of human protein kinase cAM P-activated catalytic subunit beta (PRKACB) and the 3’-UTR comprises a sequence derived from a 3’-UTR of human citrate synthase (CS);(iv) the 5’-UTR comprises a sequence derived from a 5’-UTR of human chitinase-1 (CHIT1) and the 3’-UTR comprises a sequence derived from a 3’-UTR of human chitinase-1 (CHIT1), wherein the coding sequence is not derived from human chitinase-1 (CHIT1);(v) the 5’-UTR comprises a sequence derived from a 5’-UTR of human glutamic- oxaloacetic transaminase 1 (G0T1) and the 3’-UTR comprises a sequence derived from a 3’- UTR of human citrate synthase (CS); or(vi) the 5’-UTR comprises a sequence derived from a 5’-UTR of human glutamic- oxaloacetic transaminase 1 (GOT1) and the 3’-UTR comprises a sequence derived from a 3’- UTR of human chitinase-1 (CHIT1).

2. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is a deoxyribonucleic (DNA) molecule and (i) the 5’-UTR comprises the sequence of SEQ ID NO: 1 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 9 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (ii) the 5’-UTR comprises the sequence of SEQ ID NO: 3 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 11 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (iii) the 5’-UTR comprises the sequence of SEQ ID NO: 3 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 9 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (iv) the 5’-UTR comprises the sequence of SEQ ID NO: 1 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises thesequence of SEQ ID NO: 11 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (v) the 5’-UTR comprises the sequence of SEQ ID NO: 5 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 9 or a sequence at least 80%, 85%, 90% or 95% identical thereto; or (vi) the 5’-UTR comprises the sequence of SEQ ID NO: 5 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 11 or a sequence at least 80%, 85%, 90% or 95% identical thereto.

3. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is a ribonucleic acid molecule and (i) the 5’-UTR comprises the sequence of SEQ ID NO: 2 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 10 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (ii) the 5’- UTR comprises the sequence of SEQ ID NO: 4 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 12 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (iii) the 5’-UTR comprises the sequence of SEQ ID NO: 4 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 10 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (iv) the 5’-UTR comprises the sequence of SEQ ID NO: 2 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 12 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (v) the 5’- UTR comprises the sequence of SEQ ID NO: 6 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 10 or a sequence at least 80%, 85%, 90% or 95% identical thereto; or (vi) the 5’-UTR comprises the sequence of SEQ ID NO: 6 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3’-UTR comprises the sequence of SEQ ID NO: 12 or a sequence at least 80%, 85%, 90% or 95% identical thereto.

4. The nucleic acid molecule of claim 1 or claim 3, wherein the 5’-UTR comprises or consists of the sequence of SEQ ID NO: 2 and the 3’-UTR comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.

5. The molecule of any preceding claim, wherein the coding sequence comprises a sequence encoding a therapeutic protein or peptide.

6. The molecule of claim 5, wherein the therapeutic protein or peptide is a wild-type sequence of a human protein.

7. The molecule of claim 5 or claim 6, wherein the therapeutic protein or peptide is an antibody, or antigen binding fragment thereof.

8. The molecule of claim 5, wherein the therapeutic protein or peptide comprises a disease-associated antigen (DAA).

9. The molecule of claim 8, wherein the disease-associated antigen is a tumour- associated antigen, a viral antigen or a bacterial antigen.

10. The molecule of claim 9, wherein the tumour-associated antigen is not expressed in normal tissue or is mutated in tumour cells.

11. The molecule of any one of claims 1 and 3 to 10, wherein the nucleic acid molecule is mRNA.

12. The molecule according to any one of claims 8 to 11 , wherein the coding sequence further encodes a multimerization unit (MU).

13. The molecule of claim 12, wherein the multimerization unit (MU) is ferritin.

14. The molecule of claim 12 or claim 13, wherein the coding sequence further encodes a linker, optionally a glycine-serine linker.

15. The molecule of claim 14, wherein the linker is encoded between the DAA and the MU, thereby encoding a DAA-MU fusion protein.

16. The molecule according to any one of claims 9 to 15, wherein the viral antigen is a coronavirus antigen.

17. The molecule according to claim 16, wherein the coronavirus is selected from SARS- CoV-1 and / or SARS-CoV-2.

18. The molecule according to claim 16 or claim 17, wherein the coronavirus antigen is a coronavirus spike (S) protein, or antigenic fragment thereof.

19. The molecule according to claim 18, wherein the S protein is stabilised in a pre-fusion conformation.

20. The molecule according to claim 18 or claim 19, wherein the S protein comprises K986P and V987P mutations.

21. The molecule according to claim 18, wherein the antigenic fragment thereof is a receptor binding domain (RBD).

22. A composition comprising a first nucleic acid vector comprising the molecule of any one of claims 11 to 21 , wherein the disease-associated antigen is a Wuhan variant Spike (S) Protein or a Delta variant Spike (S) Protein.

23. The composition of claim 22, wherein the first nucleic acid vector comprises the coding sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 25.

24. The composition of claim 22 or 23, further comprising a second nucleic acid vector comprising a molecule of any one of claims 11 to 21 , wherein the disease-associated antigen is an Omicron variant Spike (S) Protein, optionally variant BA.2, BA.4 / 5 or XBB.1.5.

25. The composition of claim 24, wherein the second nucleic acid vector comprises the coding sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 42.

26. The composition of any one of claims 22 to 25, wherein the first and / or second nucleic acid vector(s) comprises a polyadenylation sequence comprising between 60 and 100 adenine nucleotides.

27. The composition of any one of claims 22 to 26, wherein the first and / or second nucleic acid vector(s) comprises N1 -methylpseudouridine at 80% to 100% of uridine positions.

28. The composition of any one of claims 22 to 27, wherein the first and / or nucleic acid vector(s) comprise 5’-cap structure, optionally a cap 1 structure.

29. The composition of any one of claims 22 to 28, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5’-UTR sequence set forth in SEQ ID NO: 19 and a 3’-UTR sequence set forth in SEQ ID NO: 21 .

30. A composition comprising a nucleic acid vector comprising the molecule of any one of claims 11 to 21 , wherein the disease-associated antigen is an Omicron variant Spike (S) Protein, optionally variant BA.2, BA.4 / 5 or XBB.1.5.

31. The composition of claim 30, wherein the nucleic acid vector comprises the coding sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 42.

32. The composition of any one of claims 30 to 31 , wherein the nucleic acid vector comprises a polyadenylation sequence comprising between 60 and 100 adenine nucleotides.

33. The composition of any one of claims 30 to 32, wherein the first and / or second nucleic acid vector(s) comprises N1 -methylpseudouridine at 80% to 100% of uridine positions.

34. The composition of any one of claims 30 to 33, wherein the first and / or nucleic acid vector(s) comprise 5’-cap structure, optionally a cap 1 structure.

35. The composition of any one of claims 30 to 34, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5’-UTR sequence set forth in SEQ ID NO: 19 and a 3’-UTR sequence set forth in SEQ ID NO: 21 .

36. The molecule or composition according to any one of claims 11 to 35, wherein the nucleic acid vectors are formulated in lipid nanoparticles (LNPs).

37. The composition or nucleic acid molecule of any preceding claim for use in medicine.

38. A vaccine comprising the nucleic acid molecule or composition according to any preceding claim.

39. The composition or vaccine according to any one of claims 22 to 38, for use in a method of preventing and / or treating an infectious disease, optionally a disease caused by a coronavirus.

40. A method of preventing and / or treating an infectious disease in a subject, the method comprising administering an effective amount of the composition or vaccine according to any one of claims 22 to 38 to the subject optionally a disease caused by a coronavirus.41 . A method of vaccinating a subject against an infectious disease, optionally a disease caused by a coronavirus, comprising administering an effective amount of the composition or vaccine according to any one of claims 22 to 38.

42. A nucleic acid vector comprising a 5’ cap 1 structure, a 5’-UTR sequence derived from a 5’-UTR of human chitinase-1 (CHIT1), a coding sequence encoding a Wuhan variant Spike (S) Protein fused with a ferritin sequence or a Delta variant Spike (S) Protein fused with a ferritin sequence and a 3’-UTR sequence derived from a 3’-UTR of human citrate synthase (CS) and a polyadenylation sequence comprising between 70 and 90 adenine nucleotides.

43. A nucleic acid vector comprising a 5’ cap 1 structure, a 5’-UTR sequence derived from a 5’-UTR of human chitinase-1 (CHIT1), a coding sequence encoding an Omicron variant BA.2, BA.4 / 5 or XBB.1.5 Spike (S) Protein fused with a ferritin sequence and a 3’-UTR sequence derived from a 3’-UTR of human citrate synthase (CS) and a polyadenylation sequence comprising between 70 and 90 adenine nucleotides.

44. A composition comprising the nucleic acid vector of claim 42 and the nucleic acid vector of claim 43.

45. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 36.

46. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 37.

47. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 38.

48. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 43.

49. A composition comprising the vector of claim 45 and the vector of claim 46.

50. A composition comprising the vector of claim 45 or claim 46 and the vector of claim 47 or claim 48.51 A nucleic acid vector comprising a coding sequence that encodes for a polypeptide having the sequence set forth in SEQ ID NO: 39.

52. A nucleic acid vector comprising a coding sequence that encodes for a polypeptide having the sequence set forth in SEQ ID NO: 40.

53. A nucleic acid vector comprising a coding sequence that encodes for a polypeptide having the sequence set forth in SEQ ID NO:

41.

54. A nucleic acid vector comprising a coding sequence that encodes for a polypeptide having the sequence set forth in SEQ ID NO: 44.

55. The nucleic acid vector according to any of claims 51 to 54, further comprising a 5’- UTR and a 3’-UTR as set out in any of claims 1-4 or 29.

56. The nucleic acid vector according to any of claims 51 to 55, which is an mRNA.

57. The nucleic acid vector of claim 56, wherein the mRNA comprises any of the features of claims 26 to 28.

58. A composition comprising the vector of claim 51 and the vector of claim 52.

59. A composition comprising the vector of claim 51 or claim 52 and the vector of claim 53 or claim 54.

Images