ARTIFICIAL NUCLEIC ACID MOLECULES

Abstract

The invention relates to an artificial nucleic acid molecule comprising at least one open reading frame and at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, the invention relates to the use of a 3′-UTR element comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene for the stabilization and/or prolongation of protein expression from a nucleic acid sequence comprising such 3′-UTR element.

Claims

1. An artificial nucleic acid molecule comprising a. at least one open reading frame (ORF); and b. at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.

2. The artificial nucleic acid molecule according to claim 1, wherein the open reading frame (ORF) does not encode a reporter gene or is not derived from a reporter gene, wherein the reporter gene is preferably not selected from the group consisting of light emitting proteins, preferably not luciferase, fluorescent proteins, preferably not red, blue or green fluorescent proteins; enzymatic reporters; the lacZ gene from E. coli (beta-galactosidase); alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP); chloramphenicol acetyl transferase (CAT); hormones and cytokines.

3. The artificial nucleic acid molecule according to claim 1 or 2, wherein the open reading frame (ORF) and the 3′-UTR element are heterologous to each other.

4. The artificial nucleic acid molecule according to any one of claims 1 to 3, wherein the open reading frame (ORF) does not encode a FIG4 gene or is not derived from a FIG4 gene, preferably not an eukaryotic FIG4 gene, more preferably not a mammalian FIG4 gene, most preferably not a human FIG4 gene.

5. The artificial nucleic acid molecule according to any one of claims 1 to 4, wherein the at least one 3′-UTR element stabilizes/prolongs protein production from said artificial nucleic acid molecule.

6. The artificial nucleic acid molecule according to any one of claims 1 to 5, wherein the at least one 3′-UTR element comprises a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof.

7. The artificial nucleic acid molecule according to any one of claims 1-6, wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the at least one 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2.

8. The artificial nucleic acid molecule according to claim 7, wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

9. The artificial nucleic acid molecule according to any one of claims 1-8, wherein the at least one 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

10. The artificial nucleic acid molecule according to any one of claims 1-9 further comprising c. a poly(A) sequence and/or a polyadenylation signal.

11. The artificial nucleic acid molecule according to claim 10, wherein the poly(A) sequence or the polyadenylation signal is located 3′ of the 3′-UTR element.

12. The artificial nucleic acid molecule according to claim 10 or 11, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA.

13. The artificial nucleic acid molecule according to any one of claims 10-12, wherein the polyadenylation signal, preferably the consensus sequence NNUANA, is located less than about 50 nucleotides downstream of the 3′-end of the 3′-UTR element.

14. The artificial nucleic acid molecule according to any one of claims 10-13, wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably of about 50 to about 100 adenine nucleotides, even more preferably of about 60 to about 70 adenine nucleotides.

15. The artificial nucleic acid molecule according to any one of claims 1-14, further comprising a 5′-cap structure, a poly(C) sequence, a histone stem-loop, and/or an IRES-motif.

16. The artificial nucleic acid molecule according to any one of claims 1-15, wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 5.

17. The artificial nucleic acid molecule according to any one of claims 1-16, wherein the nucleic acid comprises an additional 5′-element, preferably a 5′-UTR, a promoter, or a 5′-UTR and a promoter containing-sequence.

18. The artificial nucleic acid molecule according to claim 17, wherein the 5′-UTR is a 5′-TOP UTR.

19. The artificial nucleic acid molecule according to any one of claims 1-18, wherein the artificial nucleic acid molecule, preferably the open reading frame, is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame.

20. The artificial nucleic acid molecule according to any one of claims 1-19, wherein the open reading frame comprises a codon-optimized region, preferably, wherein the open reading frame is codon-optimized.

21. The artificial nucleic acid molecule according to any one of claims 1-20, which is an RNA, preferably an mRNA molecule.

22. A vector comprising a. an open reading frame and/or a cloning site; and b. at least one 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.

23. The vector according to claim 22, wherein the open reading frame (ORF) does not encode a reporter gene or is not derived from a reporter gene, wherein the reporter gene is preferably not selected from the group consisting of light emitting proteins, preferably not luciferase, fluorescent proteins, preferably not red, blue or green fluorescent proteins; enzymatic reporters; the lacZ gene from E. coli (beta-galactosidase); alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP); chloramphenicol acetyl transferase (CAT); hormones and cytokines.

24. The vector according to claim 22 or 23, wherein the open reading frame (ORF) and the 3′-UTR element are heterologous to each other.

25. The artificial nucleic acid molecule according to any one of claims 22 to 24, wherein the open reading frame (ORF) does not encode a FIG4 gene or is not derived from a FIG4 gene, preferably not an eukaryotic FIG4 gene, more preferably not a mammalian FIG4 gene, most preferably not a human FIG4 gene.

26. The vector according to any one of claims 22 to 25, wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof.

27. The vector according to any one of claims 22 to 26, wherein the at least one 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the at least one 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2.

28. The vector according to claim 27, wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

29. The vector according to any one of claims 22-28, wherein the at least one 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

30. The vector according to any one of claims 22-29 further comprising c. a poly(A) sequence and/or a polyadenylation signal.

31. The vector according to claim 30, wherein the poly(A) sequence or the polyadenylation signal is located 3′ of the 3′-UTR element.

32. The vector according to claim 30 or 31, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA.

33. The vector according to any one of claims 30-32, wherein the polyadenylation signal, preferably the consensus sequence NNUANA, is located less than about 50 nucleotides downstream of the 3′-end of the 3′-UTR element.

34. The vector according to any one of claims 30-33, wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably of about 50 to about 100 adenine nucleotides, more preferably of about 60 to about 70 adenine nucleotides.

35. The vector according to any one of claims 22-34, further comprising a poly(C) sequence, a histone stem-loop, and/or an IRES-motif.

36. The vector according to any one of claims 22-35, further comprising a 5′-element, preferably a 5′-UTR, a promoter, or a 5′-UTR5′-UTR and a promoter containing-sequence.

37. The vector according to claim 36, wherein the 5′-UTR is a 5′-TOP UTR.

38. The vector according to any one of claims 22-37, which is at least partially G/C modified, preferably wherein the open reading frame is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame.

39. The vector according to any one of claims 22-38, wherein the open reading frame comprises a codon-optimized region, preferably wherein the open reading frame is codon-optimized.

40. The vector according to any one of claims 22-39, which is a DNA vector.

41. The vector according to any one of claims 22-40, which is a plasmid vector or a viral vector, preferably a plasmid vector.

42. The vector according to any one of claims 22-41, which comprises an artificial nucleic acid molecule according to any one of claims 1-21.

43. The vector according to any one of claims 22-42, which is a circular molecule.

44. The vector according to claim 43, wherein the poly(A) sequence, the poly(C) sequence, the histone stem loop or the 3′-UTR element of the coding strand is followed in 5′.fwdarw.3′ direction by a restriction site for linearization of the circular vector molecule.

45. A cell comprising the artificial nucleic acid molecule according to any one of claims 1-21 or the vector according to any one of claims 22-44.

46. The cell according to claim 45, which is a mammalian cell.

47. The cell according to claim 45 or 46, which is a cell of a mammalian subject, preferably an isolated cell of a mammalian subject, preferably of a human subject.

48. A pharmaceutical composition comprising the artificial nucleic acid molecule according to any one of claims 1-21, the vector according to any one of claims 22-44, or the cell according to any one of claims 45-47.

49. The pharmaceutical composition according to claim 48, further comprising one or more pharmaceutically acceptable vehicles, diluents and/or excipients and/or one or more adjuvants.

50. The artificial nucleic acid molecule according to any one of claims 1-21, the vector according to any one of claims 22-44, the cell according to any one of claims 45-47, or the pharmaceutical composition according to claim 48 or 49 for use as a medicament.

51. The artificial nucleic acid molecule according to any one of claims 1-21, the vector according to any one of claims 22-44, the cell according to any one of claims 45-47, or the pharmaceutical composition according to claim 48 or 49 for use as a vaccine or for use in gene therapy.

52. A method for treating or preventing a disorder comprising administering the artificial nucleic acid molecule according to any one of claims 1-21, the vector according to any one of claims 22-44, the cell according to any one of claims 45-47, or the pharmaceutical composition according to claim 48 or 49 to a subject in need thereof.

53. A method of treating or preventing a disorder comprising transfection of a cell with an artificial nucleic acid molecule according to any one of claims 1-21 or with the vector according to any one of claims 22-44.

54. The method according to claim 53, wherein transfection of a cell is performed in vitro/ex vivo and the transfected cell is administered to a subject in need thereof, preferably to a human patient.

55. The method according to claim 54, wherein the cell, which is to be transfected in vitro, is an isolated cell of the subject, preferably of the human patient.

56. The method according to any one of claims 52-55, which is a vaccination method or a gene therapy method.

57. A method for stabilizing and/or prolonging protein production from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector, the method comprising the step of associating the nucleic acid molecule, preferably the mRNA molecule or the vector, with an 3′-UTR element, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.

58. Use of an 3′-UTR element for stabilizing and/or prolonging protein production from a nucleic acid molecule, preferably from an mRNA molecule or a vector, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a FIG4 gene or from a variant of the 3′-UTR of a FIG4 gene.

59. The method according to claim 57 or the use according to claim 58, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate FIG4 gene or from a variant thereof, preferably from the 3′-UTR of a mammalian FIG4 gene or from a variant thereof, more preferably from the 3′-UTR of a primate FIG4 gene, in particular of the human FIG4 gene, or from a variant thereof, even more preferably from the 3′-UTR of the human FIG4 gene according to GenBank Accession number NM_014845.5 or from a variant thereof.

60. The method or the use according to any one of claims 57-59, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2, or wherein the 3′-UTR element comprises or consists of a fragment of a nucleic acid sequence that has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 or SEQ ID No. 2.

61. The method or the use according to claim 60, wherein the fragment exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

62. The method or the use according to any one of claims 57-61, wherein the 3′-UTR element exhibits a length of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.

63. A kit or kit of parts comprising an artificial nucleic acid molecule according to any one of claims 1-21, a vector according to any one of claims 22-44, a cell according to any one of claims 45-47, and/or a pharmaceutical composition according to claim 48 or 49.

64. The kit according to claim 63 further comprising instructions for use, cells for transfection, an adjuvant, means for administration of the pharmaceutical composition, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable solution for dissolution or dilution of the artificial nucleic acid molecule, the vector, the cells or the pharmaceutical composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0307] The following Figures, Sequences and Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0308] FIG. 1: Effect of the human FIG4 3′-UTR on luciferase expression from an artificial mRNA. Therein, the mRNA comprising the human FIG4 3′-UTR is an mRNA according to the present invention. It comprises—in 5′-to-3′-direction—a 32L4 5′-UTR5′-UTR, a GC-enriched sequence encoding Photinus pyralis luciferase, a 3′-UTR element according to SEQ ID No. 2, a poly(A) sequence having a length of 64 adenines and a poly(C) sequence of 30 cytidine nucleotides. A markedly extended protein expression from the artificial mRNA containing the human FIG4 3′-UTR corresponding to SEQ ID No. 2 is observable compared to the mRNA lacking the FIG4 3′ UTR. Data are graphed as RLU±SD (mean of relative light units±standard deviation) for triplicate transfections. RLU are summarized in Example 5.1. [0309] The following abbreviations are used: [0310] PpLuc (GC): GC-enriched mRNA sequence coding for Photinus pyralis luciferase [0311] A64: poly(A)-sequence with 64 adenylates [0312] C30: poly(C)-sequence with 30 cytidylates [0313] hSL: a histone stem-loop sequence taken from (Cakmakci, Lerner, Wagner, Zheng, & William F Marzluff, 2008. Mol. Cell. Biol. 28(3):1182-94). [0314] 32L4: 5′-UTR of human ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidine tract [0315] fig4: 3′-UTR of human fig4 (Homo sapiens FIG. 4 homolog, SAC1 lipid phosphatase domain containing (S. cerevisiae) (FIG. 4), mRNA).

[0316] FIG. 2: mRNA sequence of 32L4-PpLuc(GC)-A64-C30-hSL (SEQ ID NO: 8). [0317] The 5′-UTR is derived from human ribosomal protein Large 32 mRNA lacking the 5′ terminal oligopyrimidine tract. The PpLuc(GC) ORF is highlighted in italics.

[0318] FIG. 3: mRNA sequence of 32L4-PpLuc(GC)-fig4-A64-C30-hSL (SEQ ID NO: 7). [0319] The 3′-UTR is derived from human FIG. 4 transcript. The PpLuc(GC) ORF is highlighted in italics, the 3′ UTR is underlined.

[0320] FIG. 4: DNA sequence corresponding to mRNA PpLuc(GC)-A64-C30-hSL (SEQ ID NO: 9). [0321] The PpLuc(GC) ORF is highlighted in italics.

[0322] FIG. 5: mRNA sequence of PpLuc(GC)-fig4-A64-C30-hSL (SEQ ID NO: 10). [0323] The 3′-UTR is derived from human FIG. 4 transcript. The PpLuc(GC) ORF is highlighted in italics, the 3′ UTR is underlined.

[0324] FIG. 6: Effect of the presence of a fig4 3′-UTR on protein expression from an mRNA. Luciferase expression is shown after transfection of HeLa cells with an mRNA comprising a fig4 3′-UTR and the respective control construct without a fig4 3′-UTR.

[0325] FIGS. 7A-B: Protein expression after intradermal injection in mice. Luciferase expression has been measured after injection of an mRNA comprising a fig4 3′-UTR or an mRNA comprising an albumin7 3′-UTR, respectively. A. Time course of protein expression. B. Area under the curve (AUC).

[0326] FIG. 8: DNA sequence corresponding to mRNA RPL32-PpLuc(GC)-albumin7-A64-C30-histoneSL (SEQ ID NO: 11).

EXAMPLES

1. Preparation of DNA-Templates

[0327] A vector for in vitro transcription was used containing a T7 promoter followed by a 32L4 5′-UTR, a GC-enriched sequence coding for Photinus pyralis luciferase (Ppluc(GC)), and an A64 poly(A) sequence. The A64 poly(A) sequence is followed by C30, a histone stem-loop sequence and a restriction site used for linearization of the vector before in vitro transcription.

[0328] This vector was modified to include the 3′-UTR of the human fig4 transcript 3′ of the PpLuc ORF.

[0329] mRNAs obtained from these vectors by in vitro transcription are designated as: [0330] 32L4-PpLuc(GC)-A64-C30-hSL (FIG. 2; SEQ ID NO: 8) [0331] 32L4-PpLuc(GC)-fig4-A64-C30-hSL (FIG. 3; SEQ ID NO: 7)

[0332] A further vector for in vitro transcription was used containing a T7 promoter followed by a GC-enriched sequence encoding Photinus pyralis luciferase (Ppluc(GC)), and an A64 poly(A) sequence. The A64 poly(A) sequence is followed by C30, a histone stem-loop sequence and a restriction site used for linearization of the vector before in vitro transcription.

[0333] Also this vector was modified to include the 3′UTR of the human fig4 transcript 3′ of the PpLuc ORF.

[0334] mRNAs obtained from these vectors by in vitro transcription are designated as: [0335] PpLuc(GC)-A64-C30-hSL (FIG. 4; SEQ ID NO: 9) [0336] PpLuc(GC)-fig4-A64-C30-hSL (FIG. 5; SEQ ID NO: 10).

2. In Vitro Transcription

[0337] The DNA-template according to Example 1 was linearized and transcribed in vitro using T7 RNA polymerase. The DNA template was then digested by DNase-treatment. mRNA transcripts contained a 5′-CAP structure obtained by adding an excess of N7-Methyl-Guanosine-5′-Triphosphate-5′-Guanosine to the transcription reaction. mRNA thus obtained was purified and resuspended in water.

3. Luciferase Expression by mRNA Lipofection

[0338] Human HeLa cells were seeded in 96 well plates at a density of 1×10.sup.4 cells per well medium (RPMI 1640 medium with L-glutamine and 25 mM Hepes (Lonza, Basel, Switzerland) to which 10% FCS, 1% Pen/Strep, 1% Glutamine were added). The following day, cells were washed in Opti-MEM® I Reduced Serum Medium (Gibco, Life Technologies, Carlsbad, CA, USA) and then transfected with 12,5 ng per well of Lipofectamine2000-complexed PpLuc-encoding mRNA in Opti-MEM. Untransfected cells served as control. mRNA coding for Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (1 ng of RrLuc mRNA per well). 90 minutes after start of transfection, Opti-MEM was exchanged for medium. 24, 48, 72 hours after transfection, medium was aspirated and cells were lysed in 100 μl of Passive Lysis buffer (Promega). Lysates were stored at −80° C. until luciferase activity was measured.

4. Luciferase Measurement

[0339] Luciferase activity was measured as relative light units (RLU) in a Hidex Chameleon plate reader. The activities of Ppluc and Rrluc are measured sequentially from a single sample in a dual luciferase assay. The PpLuc activity was measured first at 2 seconds measuring time using 20 μl of lysate and 50 μl of Beetle juice (pjk GmbH). After 1500 ms delay RrLuc activity is measured with 50 μl Renilla juice (pjk GmbH).

5. Luciferase Expression by Intradermal mRNA Injection

[0340] Anaesthetized, female Balb/C mice received 4 intradermal injections per mouse (10 intradermal injections per group). Per injection 2 μg of PpLuc encoding mRNA were administered in 40 μl of 80% RiLa. 1 day, 2 days, 3 days, 4 days and 7 days after injection the anaesthetized mice are injected intraperitoneally with 150 μl of Luciferin solution (20 g/l). 10 minutes after Luciferin injection PpLuc levels are measured by optical imaging using the IVIS Lumina II System.

Results

[0341] 6.1 Protein Expression from mRNA Containing a FIG4 3′-UTR is Prolonged.

[0342] To investigate the effect of FIG4 3′-UTR protein expression from mRNA, an mRNA containing FIG4 3′-UTR (FIG. 3; SEQ ID NO: 7) was compared to a corresponding mRNA lacking the FIG4′ 3′-UTR (FIG. 2; SEQ ID NO: 8).

[0343] Human HeLa cells were transfected with Luciferase encoding mRNAs and Luciferase levels were measured 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc (see following Table 1 and FIG. 1).

TABLE-US-00004 TABLE 1 PpLuc expression normalized to RrLuc (mean RLU values are given) RLU at 24 RLU at 48 RLU at 72 mRNA hours hours hours 32L-PpLuc(GC)-A64-C30-hSL 2104506 254095 23803 32L-PpLuc(GC)-FIG4-A64- 2194153 840772 290597 C30-hSL

[0344] Luciferase was clearly expressed from mRNA lacking the FIG4 3′-UTR. The FIG4 3′-UTR significantly extended luciferase expression.

[0345] In the same manner, the expression of luciferase expressed from PpLuc(GC)-A64-C30-hSL (SEQ ID NO: 9; FIG. 4) and the expression of luciferase expressed from PpLuc(GC)-fig4-A64-C30-hSL (SEQ ID NO: 10; FIG. 5). The results are shown in Table 2 and FIG. 6.

TABLE-US-00005 TABLE 2 RLU at 24 RLU at 48 RLU at 72 mRNA hours hours hours PpLuc(GC)-A64-C30-hSL 499469 85598 13542 PpLuc(GC)-fig4-C30-hSL 505983 238912 69598

[0346] Also in the case of SEQ ID NO: 10, the FIG4 3′-UTR significantly extended luciferase expression after transfection in HeLa cells.

[0347] 6.2 Protein Expression from mRNA Containing a FIG4 3′UTR is Increased after Injection in Mice.

[0348] To investigate the effect of the FIG4 3′UTR on protein expression from mRNA, an mRNA containing the fig4 3′UTR (32L4-PpLuc(GC)-fig4-A64-C30-hSL; SEQ ID NO: 7; FIG. 3) was compared to an mRNA containing the albumin7 3′UTR (RPL32-PpLuc(GC)-albumin7-A64-C30-histoneSL; SEQ ID NO: 11; FIG. 8), the latter of which has been reported to extend the expression of a protein encoded by an associated ORF (see WO2013/143698).

[0349] Mice were injected intradermally with luciferase encoding the respective mRNAs. 1 day, 2 days, 3 days, 4 days and 7 days after injection, luciferase levels were measured. The results are shown in Table 3 and FIG. 7.

TABLE-US-00006 TABLE 3 PpLuc expression (mean RLU values are given) 32L4-PpLuc(GC)- 32L4-PpLuc(GC)- albumin7-A64-C30-hSL fig4-A64-C30-hSL Day 1 176720000 329600000 Day 2 89590000 183050000 Day 3 32020000 51960000 Day 4 12082000 36150000 Day 7 1203400 2749800

[0350] In comparison with the albumin7 3′-UTR, the fig4 3′-UTR significantly increased the expression of luciferase protein from the respective mRNA.