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) and/or at least one 5-untranslated region element (5-UTR element), wherein the at least one 3-UTR element and/or the at least one 5-UTR element prolongs and/or increases protein production from said artificial nucleic acid molecule and wherein the at least one 3-UTR element and/or the at least one 5-UTR element is derived from a stable mRNA. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, methods for identifying a 3-UTR element and/or a 5-UTR derived from a stable mRNA element are disclosed.

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), that comprises a nucleic acid sequence that is derived from the 3-UTR of a transcript of a GNAS (guanine nucleotide-binding protein G subunit alpha) gene and wherein the open reading frame is derived from a gene, which is distinct from a gene from which the at least one 3-UTR element.

2. (canceled)

3. The artificial nucleic acid molecule according to claim 1 comprising at least one 5-UTR element.

4. The artificial nucleic acid molecule according to claim 3, wherein each of the at least one open reading frame, the at least one 3-UTR element and the at least one 5-UTR element are heterologous to each other.

5-7. (canceled)

8. The artificial nucleic acid molecule according to claim 1, wherein the at least one 3-UTR element and/or the at least one comprises or consists of a nucleic acid sequence which is derived from the 3 UTR human or mouse GNAS gene.

9-11. (canceled)

12. The artificial nucleic acid molecule according to claim 3, wherein: (i) the at least one 5-UTR element is derived from a human or a murine gene selected from the group consisting of: housekeeping genes, genes coding for a membrane protein, genes involved in cellular metabolism, genes involved in transcription, translation and replication processes, genes involved in protein modification and genes involved in cell division; or (ii) the 5-UTR is not a 5 TOP UTR.

13-24. (canceled)

25. The artificial nucleic acid molecule according to claim 1, wherein the at least one 3-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 80% a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and SEQ ID NO: 116.

26. The artificial nucleic acid molecule according claim 1, wherein the at least one 5-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 80%.

27. (canceled)

28. The artificial nucleic acid molecule according to claim 1, wherein the at least one 3-UTR element and/or the at least one 5-UTR element exhibits a length of between 3 and about 500 nucleotides.

29. The artificial nucleic acid molecule according claim 1 further comprising c. a poly(A) sequence and/or a polyadenylation signal.

30-33. (canceled)

34. The artificial nucleic acid molecule according to claim 1, further comprising a 5-cap structure, a poly(C) sequence, a histone stem-loop, and/or an IRES-motif.

35. The artificial nucleic acid molecule according to claim 34, wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 34.

36. The artificial nucleic acid molecule according to claim 1, wherein the nucleic acid comprises a promoter.

37. The artificial nucleic acid molecule according to claim 1, wherein the nucleic acid comprises a 5-TOP UTR.

38-41. (canceled)

42. A vector comprising an artificial nucleic acid molecule according to claim 1.

43-46. (canceled)

47. A cell comprising the artificial nucleic acid molecule according to claim 1.

48-49. (canceled)

50. A pharmaceutical composition comprising the artificial nucleic acid molecule according to claim 1.

51-53. (canceled)

54. A method for treating or preventing a disorder comprising administering the artificial nucleic acid molecule according to claim 1 to a patient in need thereof.

55. A method of treating or preventing a disorder comprising transfection of a cell with an artificial nucleic acid molecule according to claim 1.

56-80. (canceled)

81. The artificial nucleic acid molecule according to claim 5, wherein the at least one 5-UTR element is derived from a 5-TOP UTR.

82. The artificial nucleic acid molecule according to claim 81, wherein the at least one 5-UTR element comprises a sequence of SEQ ID NO: 33.

Description

[0470] FIG. 1: shows SEQ ID NO. 35, i.e. the mRNA sequence of 32L4-PpLuc(GC)-A64-C30-hSL. (R2464). The 5-UTR is derived of human ribosomal protein Large 32 mRNA lacking the 5 terminal oligopyrimidine tract. The PpLuc(GC) ORF is highlighted in italics.

[0471] FIG. 2: shows SEQ ID NO. 36, i.e. the mRNA sequence of 32L4-PpLuc(GC)-gnas-A64-C30-hSL. (R3089). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse Gnas transcript, is underlined.

[0472] FIG. 3: shows SEQ ID NO. 37, i.e. the mRNA sequence of 32L4-PpLuc(GC)-morn2-A64-C30-hSL. (R3106). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse morn2, is underlined.

[0473] FIG. 4: shows SEQ ID NO. 38, i.e. the mRNA sequence of 32L4-PpLuc(GC)-gstm1-A64-C30-hSL. (R3107). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse gstm1, is underlined.

[0474] FIG. 5: shows SEQ ID NO. 39, i.e. the mRNA sequence of 32L4-PpLuc(GC)-ndufa1-A64-C30-hSL. (R3108). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse ndufa1, is underlined.

[0475] FIG. 6: shows SEQ ID NO. 40, i.e. the mRNA sequence of 32L4-PpLuc(GC)-cbr2-A64-C30-hSL. (R3109). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse cbr2, is underlined.

[0476] FIG. 7: shows SEQ ID NO. 41, i.e. the mRNA sequence of PpLuc(GC)-albumin7-A64-C30-hSL. (R2463). The 3-UTR is derived from human albumin with three single point mutations introduced to remove a T7 termination signal as well as a HindIII and XbaI restriction site (albumin7). The PpLuc(GC) ORF is highlighted in italics.

[0477] FIG. 8: shows SEQ ID NO. 42, i.e. the mRNA sequence of Mp68-PpLuc(GC)-albumin7-A64-C30-hSL. (R3111). The PpLuc(GC) ORF is highlighted in italics. The 5-UTR element, which is derived from mouse mp68, is underlined.

[0478] FIG. 9: shows SEQ ID NO. 43, i.e. the mRNA sequence of Ndufa4-PpLuc(GC)-albumin7-A64-C30-hSL. (R3112). The PpLuc(GC) ORF is highlighted in italics. The 5-UTR element, which is derived from mouse Ndufa4, is underlined.

[0479] FIG. 10: shows SEQ ID NO. 44, i.e. the mRNA sequence of PpLuc(GC)-A64-C30-hSL (R2462) The PpLuc(GC) ORF is highlighted in italics.

[0480] FIG. 11: shows SEQ ID NO. 45, i.e. the mRNA sequence of PpLuc(GC)-gnas-A64-C30-hSL(R3116). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse Gnas, is underlined.

[0481] FIG. 12: shows that different 3-UTR elements, namely 3-UTR elements derived from gnas, morn2, gstm1, ndufa1 and cbr2 markedly prolong protein expression from mRNA. [0482] The effect of the inventive 3-UTR elements derived from gnas, morn2, gstm1, ndufa1 and cbr2 3-UTRs on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, human HeLa were transfected with different mRNAs by lipofection. Luciferase levels were measured at different times after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h was calculated. The 3-UTRs prolong luciferase expression. Mean values from three independent experiments are shown. Values are summarized in Example 7.a.

[0483] FIG. 13: shows that different 3-UTR elements, namely 3-UTR elements derived from gnas, morn2, gstm1, ndufa1 and cbr2 markedly prolong protein expression from mRNA. [0484] The effect of the inventive 3-UTR elements derived from gnas, morn2, gstm1, ndufa1 and cbr2 3-UTRs on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, HDF (human dermal fibroblasts) cells were transfected with different mRNAs by lipofection. Luciferase levels were measured at different times after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h was calculated. The 3-UTRs prolong luciferase expression. Mean values from three independent experiments are shown. Values are summarized in Example 7.a.

[0485] FIG. 14: shows that different 5-UTR elements, namely 5-UTR elements derived from Mp68 and ndufa4 markedly increase total protein expression from mRNA. [0486] The effect of the inventive 5-UTR elements derived from Mp68 and ndufa4 on luciferase expression from mRNA was examined. To this end, human HeLa cells were transfected with different mRNAs by lipofection. Luciferase levels were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Total protein expression (area under the curve) was calculated. To compare the expression levels of the mRNAs containing the inventive 5-UTR elements to an mRNA lacking a 5-UTR, expression levels of the control construct without 5 UTR was set to 1. Mean values from three independent experiments are shown. Values are summarized in Example 7.b.

[0487] FIG. 15: shows that different 5-UTR elements, namely 5-UTR elements derived from Mp68 and ndufa4 markedly increase total protein expression from mRNA. [0488] The effect of the inventive 5-UTR elements derived from Mp68 and ndufa4 on luciferase expression from mRNA was examined. To this end, HDF cells were transfected with different mRNAs by lipofection. Luciferase levels were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Total protein expression (area under the curve) was calculated. To compare the expression levels of the mRNAs containing the inventive 5-UTR elements to an mRNA lacking a 5-UTR, expression levels of the control construct without 5 UTR was set to 1. Mean values from three independent experiments are shown. Values are summarized in Example 7.b.

[0489] FIG. 16: shows that the 3-UTR element derived from gnas markedly prolongs protein expression from mRNA. [0490] The effect of the inventive 3-UTR element derived from gnas 3-UTR on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, HDF cells were transfected with respective mRNAs by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h was calculated. The gnas 3-UTR prolongs luciferase expression. Values are summarized in Example 7.c.

[0491] FIG. 17: shows that the 3-UTR element derived from gnas markedly prolongs protein expression from mRNA. [0492] The effect of the inventive 3-UTR element derived from gnas 3-UTR on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, HeLa cells were transfected with respective mRNAs by lipofection. Luciferase levels were measured at d2 and d3 after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h was calculated. The gnas 3-UTR prolongs luciferase expression. Values are summarized in Example 7.c.

[0493] FIG. 18: shows that different 3-UTR elements, namely 3-UTR elements derived from ybx1(V2), ndufb8, and cntn1-004(V2) markedly prolong protein expression from mRNA. [0494] The effect of the inventive 3-UTR elements derived from ybx1(V2), ndufb8, and cntn1-004(V2) 3-UTRs on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, HDF cells were transfected with the different mRNAs by lipofection. Luciferase levels were measured at different times after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h was calculated. The 3-UTRs prolong luciferase expression. Values are summarized in Example 7.d.

[0495] FIG. 19: shows SEQ ID NO. 46, i.e. the mRNA sequence of 32L4-PpLuc(GC)-Ybx1-001(V2)-A64-C30-hSL (R3623) Mus musculus 3UTR with mutation T128bpG and deletion de1236-237 bp. The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse Ybx1 transcript, is underlined.

[0496] FIG. 20: shows SEQ ID NO. 47, i.e. the mRNA sequence of 32L4-PpLuc(GC)-Ndufb8-A64-C30-hSL (R3624) The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from mouse Ndufb8 transcript, is underlined.

[0497] FIG. 21: shows SEQ ID NO. 48, i.e. the mRNA sequence of 32L4-PpLuc(GC)-Cntn1-004(V2)-A64-C30-hSL (R3625)+T at pos. 30 bp, mutations G727bpT, A840bpG. The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from human Cntn1 transcript, is underlined.

[0498] FIG. 22: shows that different 3-UTR elements, namely 3-UTR elements derived from gnas, morn2, ndufa1 (Mm; Mus musculus), and NDUFA1 (Hs; Homo sapiens) markedly prolong protein expression from mRNA. The effect of the inventive 3-UTR elements derived from gnas, morn2, ndufa1 (Mm; Mus musculus), and NDUFA1 (Hs; Homo sapiens) on luciferase expression from mRNA was examined, compared to luciferase expression from mRNA lacking a 3-UTR. To this end, human Hela cells were transfected with respective mRNAs by lipofection. Luciferase levels were measured at different times after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h is calculated. The 3UTRs prolong luciferase expression. Mean values from 3 independent experiments are shown. Values are summarized in Table 8.

[0499] FIG. 23: shows that different 5-UTR elements, namely 5-UTR elements derived from Mp68 and ndufa4, markedly increase total protein expression from mRNA. The effect of the inventive 5-UTR elements derived from Mp68 and ndufa4 on luciferase expression from mRNA was examined. To this end, human HeLa cells were transfected with different mRNAs by lipofection. Luciferase levels were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Total protein expression (area under the curve) was calculated. To compare the expression levels of the mRNAs containing the inventive 5-UTR elements to an mRNA lacking a 5-UTR, expression levels of the control construct without 5 UTR was set to 1. Mean values are shown. Values are summarized in Table 9.

[0500] FIG. 24: shows that different 5-UTR elements, namely 5-UTR elements derived from Mp68 and ndufa4, markedly increase total protein expression from mRNA. The effect of the inventive 5-UTR elements derived from Mp68 and ndufa4 on luciferase expression from mRNA was examined. To this end, human Hela cells were transfected with different mRNAs by lipofection. 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. Total protein expression (area under the curve) was calculated. To compare the expression levels of the mRNAs containing the inventive 5-UTR elements to an mRNA lacking a 5-UTR, expression levels of the control construct without 5 UTR was set to 1. Mean values are shown. Values are summarized in Table 9.

[0501] FIG. 25: shows SEQ ID NO. 383, i.e. the mRNA sequence of 32L4-PpLuc(GC)-A64-C30-hSL (R2462). The PpLuc(GC) ORF is highlighted in italics.

[0502] FIG. 26: shows SEQ ID NO. 384, i.e. the mRNA sequence of PpLuc(GC)-morn2-A64-C30-hSL. (R3948). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from murine morn2 is underlined.

[0503] FIG. 27: shows SEQ ID NO. 385, i.e. the mRNA sequence of PpLuc(GC)-ndufa1-A64-C30-hSL. (R4043). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from murine ndufa1 is underlined.

[0504] FIG. 28: shows SEQ ID NO. 386, i.e. the mRNA sequence of PpLuc(GC)-NDFUA1-A64-C30-hSL. (R3948). The PpLuc(GC) ORF is highlighted in italics. The 3-UTR element, which is derived from human NDUFA1 is underlined.

[0505] FIG. 29: shows SEQ ID NO. 387, i.e. the mRNA sequence of Mp68-PpLuc(GC)-A64-C30-hSL. (R3954). The PpLuc(GC) ORF is highlighted in italics. The 5-UTR element, which is derived from murine mp68 is underlined.

[0506] FIG. 30: shows SEQ ID NO. 388, i.e. the mRNA sequence of Ndufa4-PpLuc(GC)-A64-C30-hSL. (R3951). The PpLuc(GC) ORF is highlighted in italics. The 5-UTR element, which is derived from murine ndufa4 is underlined.

EXAMPLES

[0507] 1. Identification of 3-Untranslated Region Elements (3-UTR Elements) and/or 5-Untranslated Region Elements (5-UTR Elements) Prolonging and/or Increasing Protein Production:

[0508] mRNA decay in different human and murine cell types was assessed by pulse-chase methodology. To this end, three different human cell types (HeLa, HDF and U-937) and three different mouse cell types (NIH3T3, JAWSII and L929) were plated over night in their respective medium: HeLa, U-937, L929 in RPMI medium, JAWSII und NIH3T3 in DMEM and HDF in Fibroblast Growth Medium 2. The cells were incubated for 3 h with the respective medium containing 200 M 4-thiouridine (4sU) for labelling of newly synthesized RNA (pulse). After incubation (labelling), cells are washed once and the medium was replaced by fresh medium supplemented with 2 mM Uridine (chase). Cells were incubated further for 3 h (1st point in time) or 15 h (2.sup.nd point in time) before harvesting.

[0509] Accordingly, cells were harvested 3 h (1st point in time) and 15 h (2.sup.nd point in time) after end of labelling. The total RNA was isolated from these cells using RNeasy Mini Kit (Qiagen).

[0510] HPDP-Biotin (EZ-Link Biotin-HPDP, Thermo Scientific; pyridyldithiol-activated, sulfhydryl-reactive biotinylation reagent that conjugates via a cleavable (reversible) disulfide bond) was then incubated with the total RNA in order to extract the 4-thiouridine (4sU)-labelled RNA. HPDP-Biotin specifically reacts with the reduced thiols (SH) in the 4-thiouridine (4sU)-labelled RNA to form reversible disulfide bonds. The biotinylated RNA was ultrafiltrated using an Amicon-30 device, incubated with streptavidin-coupled dynabeads (Life Technologies) and recovered from streptavidin by DTT. Subsequently, the RNA was purified using RNeasy Mini Kit. For each cell line 3 independent experiments were performed.

[0511] The extracted 4sU-labelled RNA was used in a micro array analysis in order to determine the transcript levels of a large variety of mRNA species (i.e. the amounts of the mRNA species) present at a first point in time (3 h after labelling) and the transcript levels of a large variety of mRNA species (i.e. the amounts of the mRNA species) present at a second point in time (15 h after labelling). Affymetrix Human Gene 1.0 ST and Affymetrix Mouse Gene 1.0 ST micro arrays were used. Affymetrix Human Gene 1.0 ST comprises 36079 mRNA species. Affymetrix Mouse Gene 1.0 ST comprises 26166 mRNA species.

[0512] Since these micro arrays provide a whole transcript coverage, i.e. they provide a complete expression profile of mRNA, the ratio of the transcript level of a certain mRNA species at the second point in time to the transcript level of the same mRNA species at the first point in time was accordingly determined for a large number of mRNA species. The ratios thus reflect the x-fold transcript level of the mRNA species (shown as Gene Symbol) at the second point in time as compared to the first point in time.

[0513] The results from these experiments are shown in Tables 1-3 below. Each of the Tables 1-3 shows a ranking of the most stable mRNA species, i.e. according to the ratio of the transcript level of this mRNA species at the second point in time to the transcript level of this mRNA species at the first point in time (Table 1: combined analysis of human cell types (HeLa, HDF and U-937); Table 2: combined analysis of mouse cell lines (NIH3T3, JAWSII and L929); Table 3: human cell line HDF (human dermal fibroblasts)). Such mRNA species were considered as most stable mRNA species, which show a value for the ratio of the transcript level at the first point in time/the transcript level at the second point in time of at least 0.549943138 (approximately 55%; Table 1), 0.676314425 (approximately 68%, Table 2) or 0.8033973 (approximately 80%, Table 3).

[0514] Furthermore, the relationship of the ratio of a certain mRNA species to the average ratio (i.e. the 5 average of the ratios of all mRNA species determined, which is shown in the Tables as Average of the ratio) was calculated and given as % of average.

TABLE-US-00009 TABLE 1 stable mRNAs resulting from the combined analysis of human cell types (HeLa, HDF and U-937) with the Affymetrix Human Gene 1.0 ST micro array. 113 mRNA species of the 36079 mRNA species on the micro array were selected as most stable mRNA species. This corresponds to 0.31% of the mRNA species present on the micro array. Ratio of the transcript level at the 2nd time to the transcript level at Average of % of Gene symbol the 1st time the ratio average LTA4H 0.982490359 0.258826017 379.5948991 SLC38A6 0.953694877 368.4694789 DECR1 0.927429689 358.3216631 PIGK 0.875178367 338.1338462 FAM175A 0.849392515 328.1712266 PHYH 0.827905031 319.8693239 NT5DC1 0.815986179 315.2643572 TBC1D19 0.805960687 311.3909086 PIGB 0.805108608 311.0616997 ALG6 0.804875859 310.9717748 CRYZ 0.797694475 308.1971756 BRP44L 0.796150905 307.6008021 ACADSB 0.792385554 306.1460216 SUPT3H 0.792305264 306.1150005 TMEM14A 0.792128439 306.0466827 GRAMD1C 0.78766459 304.3220303 C11orf80 0.778391775 300.739386 C9orf46 0.776061355 299.8390053 ANXA4 0.765663559 295.8217134 RAB7A 0.757621668 292.7146492 TBCK 0.753324047 291.0542204 AGA 0.751782245 290.4585303 IFI6 0.742389518 286.829557 C2orf34 0.737633511 284.9920263 TPK1 0.731359535 282.5680135 ALDH6A1 0.731062569 282.4532776 AGTPBP1 0.725606511 280.3452757 CCDC53 0.725535697 280.3179158 LRRC28 0.722761729 279.2461657 MBNL3 0.716905277 276.9834674 CCDC109B 0.713320794 275.5985668 PUS10 0.70905743 273.9513739 CCDC104 0.706185858 272.8419137 CASP1 0.699081435 270.0970494 SNX14 0.689529842 266.4066965 SKAP2 0.686417578 265.2042424 NDUFB6 0.683568924 264.1036366 EFHA1 0.680321463 262.8489478 BCKDHB 0.679714289 262.6143601 BBS2 0.677825758 261.8847077 LMBRD1 0.676629332 261.4224565 ITGA6 0.660264393 255.0996998 HERC5 0.654495807 252.8709496 HADHB 0.651220796 251.6056164 MCCC2 0.650460461 251.3118537 CAT 0.647218183 250.0591672 ANAPC4 0.646761056 249.8825517 PCCB 0.641145931 247.7130926 PHKB 0.639806797 247.1957046 ABCB7 0.639415266 247.0444329 PGCP 0.636830107 246.0456309 GPD2 0.63484437 245.2784217 TMEM38B 0.634688463 245.2181856 NFU1 0.63202654 244.1897253 OMA1 0.631592924 244.0221934 LOC128322 0.630915328 243.7603974 NUBPL 0.627949735 242.6146113 LANCL1 0.627743069 242.5347636 HHLA3 0.62723119 242.3369941 PIR 0.625871255 241.8115696 ACAA2 0.624054189 241.1095284 CTBS 0.621758355 240.22251 GSTM4 0.618559637 238.9866536 ALG8 0.617468882 238.5652294 ACTR10 0.614629804 237.4683237 PIGF 0.612863425 236.7858655 MGST3 0.607459796 234.6981198 SCP2 0.604745109 233.6492735 HPRT1 0.604586436 233.5879689 ACSF2 0.603568827 233.1948052 VPS13A 0.60079506 232.1231332 CTH 0.598492068 231.2333494 NXT2 0.597938464 231.0194589 MGST2 0.596121512 230.3174615 C11orf67 0.59596274 230.2561181 PCCA 0.595915054 230.2376943 GLMN 0.594596168 229.7281295 DHRS1 0.594391166 229.6489249 PON2 0.594025719 229.5077308 NME7 0.593140523 229.1657265 ETFDH 0.59290737 229.0756456 ALG13 0.591519568 228.5394547 DDX60 0.590567649 228.1716714 DYNC2LI1 0.590400874 228.1072359 VPS8 0.586233686 226.4972016 ITFG1 0.585791975 226.3265424 CDK5 0.584517109 225.8339853 C1orf112 0.58415003 225.6921603 IFT52 0.579757269 223.9949738 CLYBL 0.577777391 223.230028 FAM114A2 0.575975081 222.533688 NUDT7 0.575398988 222.3111085 AKD1 0.57519887 222.233791 MAGED2 0.575157132 222.217665 HRSP12 0.574805797 222.0819235 STX8 0.573508131 221.5805571 ACAT1 0.569067306 219.8648003 IFT74 0.568627867 219.695019 KIFAP3 0.567709483 219.3401921 CAPN1 0.567537877 219.2738902 COX11 0.566354405 218.8166442 GLT8D4 0.566035014 218.6932442 HACL1 0.56371793 217.7980159 IFT88 0.562663344 217.3905661 NDUFB3 0.561240987 216.8410243 ANO10 0.561096127 216.7850564 ARL6 0.560155258 216.4215424 LPCAT3 0.559730076 216.2572689 ABCD3 0.55747212 215.3848853 COPG2 0.557180095 215.2720583 MIPEP 0.554396343 214.1965281 LEPR 0.551799358 213.1931572 C2orf76 0.549943138 212.4759882

TABLE-US-00010 TABLE 2 stable mRNAs resulting from the combined analysis of mouse cell lines (NIH3T3, JAWSII and L929) with the Affymetrix Mouse Gene 1.0 ST micro array: 99 mRNA species of the 26166 mRNA species on the micro array were selected as the most stable mRNA species. This corresponds to 0.38% of the mRNA species present on the micro array. Ratio of the transcript level at the 2nd time to the transcript level at Average of ene symbol the 1st time the ratio % of average Ndufa1 1.571557917 0.209425963 750.4121719 Atp5e 1.444730129 689.8524465 Gstm5 1.436992822 686.1579154 Uqcr11 1.221605816 583.3115431 Ifi27l2a 1.203811772 574.8149632 Cbr2 1.162403907 555.0428852 Anapc13 1.153679871 550.8771953 Atp5l 1.126858713 538.0702074 Tmsb10 1.048459674 500.6350022 Nenf 1.045891853 499.4088786 Ndufa7 1.03898238 496.1096349 Atp5k 1.03623698 494.7987179 1110008P14Rik 1.029513775 491.5884162 Cox4i1 0.991815573 473.5876865 Cox6a1 0.991620272 473.4944312 Ndufs6 0.989419978 472.4438002 Sec61b 0.984420709 470.0566705 Romo1 0.981642576 468.7301241 Gnas 0.969128675 462.7547898 Snrpd2 0.962862199 459.7625743 Mgst3 0.96060161 458.6831531 Aldh2 0.949761281 453.5069425 2010107E04Rik 0.933570825 445.776069 Ssr4 0.930263069 444.1966294 Myl6 0.920572238 439.5692993 Prdx4 0.914830854 436.8278128 Ubl5 0.902505176 430.9423544 1110001J03Rik 0.888041155 424.0358468 Ndufa13 0.881735594 421.0249684 Ndufa3 0.880861551 420.6076163 Gstp2 0.87970004 420.0529997 Tmem160 0.878001416 419.2419142 Ergic3 0.87481135 417.7186716 Pgcp 0.870441149 415.6319192 Slpi 0.868909664 414.9006418 Myeov2 0.868175997 414.5503186 Ndufa4 0.862009116 411.6056594 Ndufs5 0.857586364 409.4938143 Gstm1 0.856672742 409.0575637 1810027O10Rik 0.855929863 408.7028424 Atp5o 0.848957424 405.3735324 Shfm1 0.841951399 402.0281856 Tspo 0.840567742 401.3674952 S100a6 0.840163495 401.1744691 Taldo1 0.8400757 401.1325475 Bloc1s1 0.838838894 400.541978 Hexa 0.826597959 394.6969835 Ndufb11 0.821601877 392.311376 Map1lc3a 0.816696063 389.968871 Morn2 0.810862522 387.18338 Gpx4 0.808459051 386.0357329 Mif 0.804105552 383.9569558 Cox6b1 0.803409855 383.6247633 2900010J23Rik 0.802900813 383.3816981 Sec61g 0.797138268 380.6301077 2900010M23Rik 0.793618387 378.9493795 Anapc5 0.793224505 378.7613023 Mars2 0.787395376 375.9779182 Phpt1 0.785668786 375.153479 Ndufb8 0.784300334 374.5000492 Pfdn5 0.779021933 371.9796349 Arpc3 0.77876305 371.8560197 Ndufb7 0.774103875 369.6312833 Atp5h 0.772255845 368.7488573 Mrpl23 0.77034041 367.834245 Tomm6 0.75481818 360.4224467 Mtch1 0.752594518 359.3606576 Pcbd2 0.752256847 359.199421 Ecm1 0.752254099 359.1981094 Hrsp12 0.749135357 357.708923 Mecr 0.746269148 356.3403207 Uqcrq 0.734462177 350.7025426 Gstm3 0.733839044 350.4049993 Lsm4 0.732100345 349.5747779 Park7 0.7307842 348.9463242 Usmg5 0.724562823 345.9756436 Cox8a 0.720194618 343.8898445 Ly6c1 0.716087602 341.9287619 Cox7b 0.713519017 340.7022736 Ppib 0.706106711 337.1629288 Bag1 0.70488561 336.5798584 S100a4 0.701675201 335.046902 Bcap31 0.700846929 334.6514056 Tecr 0.699592215 334.0522852 Rabac1 0.699161282 333.8465165 Robld3 0.694068018 331.4145049 Sod1 0.691852987 330.356837 Nedd8 0.691415017 330.1477083 Higd2a 0.689498548 329.2326025 Trappc6a 0.688046277 328.5391491 Ldhb 0.686084572 327.6024437 Nme2 0.685974394 327.5498339 Snrpg 0.684247073 326.7250454 Ndufa2 0.683350661 326.2970129 Serf1 0.681148053 325.2452768 Oaz1 0.681139695 325.2412861 Ybx1 0.678927132 324.1847964 Sepp1 0.677551422 323.5279009 Gaa 0.676314425 322.9372402

TABLE-US-00011 TABLE 3 stable mRNAs resulting from the analysis of the human cell line HDF (human dermal fibroblasts) with the Affymetrix Human Gene 1.0 ST micro array: 46 mRNA species of the 36079 mRNA species on the micro array were selected as the most stable mRNA species. This corresponds to 0.13% of the mRNA species present on the micro array. Ratio of the transcript level at the 2nd time to the transcript level at Average of Gene symbol the 1st time the ratio % of average ABCA6 2.062835692 0.278262352 741.3276273 LY96 1.719983635 618.1158256 CROT 1.422424006 511.1809038 ENPP5 1.315849211 472.880791 SERPINB7 1.12288882 403.5360196 TCP11L2 1.103519648 396.5752607 IRAK1BP1 1.05490107 379.1030521 CDKL2 1.042002646 374.4677057 GHR 1.039327135 373.5061992 KIAA1107 1.020519239 366.7471477 RPS6KA6 1.017695602 365.7324085 CLGN 1.007943464 362.2277524 TMEM45A 1.006063873 361.5522781 TBC1D8B 0.979626826 352.0515148 ACP6 0.964241225 346.5223439 RP6-213H19.1 0.960702414 345.2505905 C11orf74 0.960086216 345.0291458 SNRPN 0.939315038 337.5645433 GLRB 0.923441342 331.8599644 HERC6 0.919865006 330.5747254 CFH 0.908835974 326.6111879 GALC 0.90862766 326.5363257 PDE1A 0.908445187 326.4707497 GSTM5 0.902862912 324.4646303 CADPS2 0.89753131 322.5485959 AASS 0.894768872 321.5558503 TRIM6-TRIM34 0.892150571 320.6149031 SEPP1 0.891344657 320.3252795 PDE5A 0.890221551 319.9216656 SATB1 0.885139895 318.0954552 CCPG1 0.88148167 316.7807873 CNTN1 0.87246423 313.5401621 LMBRD2 0.871500964 313.1939903 TLR3 0.86777981 311.8567077 BCAT1 0.864255836 310.5902863 TOM1L1 0.86240499 309.925142 SLC35A1 0.857201353 308.055095 GLYATL2 0.85132258 305.9424223 STAT4 0.840572034 302.0789653 GULP1 0.839518351 301.7003001 EHHADH 0.82971807 298.1783427 NBEAL1 0.82554089 296.6771768 KIAA1598 0.820341324 294.8085928 HFE 0.815037603 292.9025779 KIAA1324L 0.808279102 290.4737547 MANSC1 0.8033973 288.7193664
2. Cloning of 5- and 3-UTR Elements of Stably Expressed mRNAs:

[0515] The nucleotide sequence of the 5- and/or 3-UTRs of the mRNA species shown in Table 1-3 were determined by data base search and amplified by PCR or synthesized by oligo annealing. The resulting PCR fragments were cloned into a vector as described in detail in Example 3 below. 5-UTR elements were cloned into the vector PpLuc(GC)-albumin7-A64-C30-hSL (SEQ ID NO. 41, FIG. 7); and 3-UTR elements were cloned into the vector 32L4-PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 35, FIG. 1) or into the vector PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 44, FIG. 10).

3. Preparation of DNA-Templates

[0516] A vector for in vitro transcription was constructed containing a T7 promoter and a GC-enriched sequence coding for Photinus pyralis luciferase (PpLuc(GC)). An A64 poly(A) sequence, followed by C30 and a histone stem-loop sequence, was inserted 3 of PpLuc(GC). The histone stem-loop sequence was followed by a restriction site used for linearization of the vector before in vitro transcription.

[0517] To investigate the effect of different 3-UTR elements on protein expression, a vector as described above was used (control) and this vector was modified to include a 3-UTR element of interest. Alternatively, a vector was constructed as described above, whereby the 5 untranslated region (5-UTR) of 32L4 (ribosomal protein Large 32) was inserted 5 of PpLuc(GC). This vector was then modified to include either different 3-UTR elements or no 3-UTR (control).

[0518] Particularly, the following mRNAs were obtained from these vectors accordingly by in vitro transcription (the mRNA sequences are depicted in FIGS. 1 to 6, FIGS. 10, 11 and FIGS. 19 to 21):

32L4-PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 35, FIG. 1);

[0519] 32L4-PpLuc(GC)-gnas-A64-C30-hSL (SEQ ID NO. 36, FIG. 2);
32L4-PpLuc(GC)-morn2-A64-C30-hSL (SEQ ID NO. 37, FIG. 3);
32L4-PpLuc(GC)-gstm1-A64-C30-hSL (SEQ ID NO. 38, FIG. 4);
32L4-PpLuc(GC)-ndufa1-A64-C30-hSL (SEQ ID NO. 39, FIG. 5);
32L4-PpLuc(GC)-cbr2-A64-C30-hSL (SEQ ID NO. 40, FIG. 6);

PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 44, FIG. 10);

[0520] PpLuc(GC)-gnas-A64-C30-hSL (SEQ ID NO. 45, FIG. 11);

32L4-PpLuc(GC)-Ybx1(V2)-A64-C30-hSL (SEQ ID NO. 46, FIG. 19);

32L4-PpLuc(GC)-Ndufb8-A64-C30-hSL (SEQ ID NO. 47, FIG. 20); and

32L4-PpLuc(GC)-Cntn1-004(V2)-A64-C30-hSL (SEQ ID NO. 48, FIG. 21).

[0521] An alternative sequence for the construct 32L4-PpLuc(GC)-A64-C30-hSL is shown in FIG. 25 (SEQ ID NO. 383). However, SEQ ID NO. 35, FIG. 1 was used in the Examples as described herein and is, thus, preferred for the construct 32L4-PpLuc(GC)-A64-C30-hSL.

[0522] To investigate the effect of different 5-UTR elements on protein expression, a vector was constructed as described above, whereby the 3 untranslated region (3-UTR) of albumin7 (3-UTR of human albumin with three single point mutations introduced to remove a T7 termination signal as well as a HindIII and XbaI restriction site) was inserted 3 of PpLuc(GC). This vector was modified to include either different 5-UTR elements or no 5-UTR (control).

[0523] Particularly, the following mRNAs were obtained from these vectors accordingly by in vitro transcription (the mRNA sequences are depicted in FIGS. 7 to 9):

PpLuc(GC)-albumin7-A64-C30-hSL (SEQ ID NO. 41, FIG. 7);
Mp68-PpLuc(GC)-albumin7-A64-C30-hSL (SEQ ID NO. 42, FIG. 8); and
Ndufa4-PpLuc(GC)-albumin7-A64-C30-hSL (SEQ ID NO. 43, FIG. 9);

4. In Vitro Transcription

[0524] The DNA templates according to Example 2 and 3 were linearized and transcribed in vitro using T7-RNA polymerase. The DNA templates were 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.

5. Luciferase Expression by mRNA Lipofection

[0525] Human dermal fibroblasts (HDF) and HeLa cells were seeded in 96 well plates at a density of 110.sup.4 cells per well. The following day, cells were washed in Opti-MEM 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. 6, 24, 48, 72 hours after transfection, medium was aspirated and cells were lysed in 100 l of Passive Lysis buffer (Promega). Lysates were stored at 80 C. until luciferase activity was measured.

6. Luciferase Measurement

[0526] 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 l of lysate and 50 l of Beetle juice (pjk GmbH). After 1500 ms delay RrLuc activity is measured with 50 l Renilla juice (pjk GmbH).

7. Results

[0527] a. Protein Expression from mRNA Containing 3-UTR Elements According to the Invention is Increased and/or Prolonged.

[0528] To investigate the effect of various 3-UTR elements on protein expression from mRNA, mRNAs containing different 3-UTR elements were compared to an mRNA lacking a 3-UTR.

[0529] Human HeLa and HDF cells were transfected with Luciferase encoding mRNAs and Luciferase levels (in RLU) were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24h were set to 100% and relative expression to 24 h is calculated (see following Table 4 and FIGS. 12 (HeLa cells) and 13 (HDF cells)).

TABLE-US-00012 TABLE 4 HeLa HDF mRNA 24 h 48 h 72 h 24 h 48 h 72 h 32L4-PpLuc(GC)-A64-C30-hSL 100 12.3 2.7 100 34.8 10.9 32L4-PpLuc(GC)-gnas-A64-C30-hSL 100 50.5 30.9 100 79.8 27.8 32L4-PpLuc(GC)-morn2-A64-C30-hSL 100 32.9 10.5 100 44.5 14.6 32L4-PpLuc(GC)-gstm1-A64-C30-hSL 100 24.8 7.6 100 46.5 21.4 32L4-PpLuc(GC)-ndufa1-A64-C30-hSL 100 29.4 10.6 100 41.9 13.9 32L4-PpLuc(GC)-cbr2-A64-C30-hSL 100 21.9 4.9 100 60.0 23.2

[0530] Table 4 shows relative PpLuc expression normalized to RrLuc (mean values of three independent experiments are given).

[0531] Luciferase was expressed from mRNA lacking a 3-UTR. However, the inventive 3-UTR elements gnas, morn2, gstm1, ndufa and cbr2 significantly prolonged luciferase expression.

[0532] b. Protein Expression from mRNA Containing 5-UTR Elements According to the Invention is Increased and/or Prolonged.

[0533] To investigate the effect of various 5-UTR elements on protein expression from mRNA, mRNAs containing different 5-UTRs were compared to an mRNA lacking a 5-UTR.

[0534] Human HeLa and HDF cells were transfected with Luciferase encoding mRNAs and Luciferase levels were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Total protein expression from 0 to 72 hours was calculated as the area under the curve (AUC). The levels of the control construct without 5 UTR was set to 1 (see following Table 5 and FIG. 14 (HeLa cells) and 15 (HDF cells)).

TABLE-US-00013 TABLE 5 mRNA AUC HeLa AUC HDF PpLuc(GC)-albumin7-A64-C30-hSL 1.00 1.07 Mp68-PpLuc(GC)-albumin7-A64-C30-hSL 1.79 3.03 Ndufa4-PpLuc(GC)-albumin7-A64-C30-hSL 1.92 2.83

[0535] Table 5 shows the total PpLuc expression normalized to RrLuc (mean values of three independent experiments are given).

[0536] Luciferase was expressed from mRNA lacking a 5-UTR. However, the inventive 5-UTR elements mp68 and ndufa4 significantly increased luciferase expression.

[0537] c. Protein Expression from mRNA Containing 3-UTR Elements According to the Invention is Prolonged.

[0538] To investigate the effect of various 3UTRs on protein expression from mRNA, mRNAs containing different 3UTRs were compared to an mRNA lacking a 3UTR.

[0539] Human HeLa and HDF cells were transfected with Luciferase encoding mRNAs and Luciferase levels (in RLU) were measured 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h is calculated (see following Table 6 and FIGS. 16 (HeLa cells) and 17 (HDF cells)).

TABLE-US-00014 TABLE 6 HeLa HDF mRNA 24 h 48 h 72 h 24 h 48 h 72 h PpLuc(GC)-gnas-A64-C30-hSL 100 61.1 30.3 100 53.6 34.2 PpLuc(GC)-A64-C30-hSL 100 17.1 2.7 100 29.0 12.4

[0540] Table 6 shows relative PpLuc expression normalized to RrLuc (mean values of three independent experiments are given).

[0541] d. Protein Expression from mRNA Containing 3-UTR Elements According to the Invention is Prolonged.

[0542] To investigate the effect of various 3UTRs on protein expression from mRNA, mRNAs containing different 3UTRs were compared to an mRNA lacking a 3UTR.

[0543] Human HeLa and HDF cells were transfected with Luciferase encoding mRNAs and Luciferase levels were measured 6, 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Total protein expression from 0 to 72 hours was calculated as the area under the curve (AUC). The levels of the control construct without 5 UTR was set to 1 (see following Table 7 and FIG. 18 (HDF cells) and 17 (HeLa cells)).

[0544] Human HeLa and HDF cells were transfected with Luciferase encoding mRNAs and Luciferase levels (in RLU) were measured 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc. Normalized PpLuc levels at 24 h were set to 100% and relative expression to 24 h is calculated (see following Table 7 and FIG. 18 (HDF cells)).

TABLE-US-00015 TABLE 7 HDF mRNA 24 h 48 h 72 h 32L4-PpLuc(GC)-Ybx1-001(V2)-A64-C30-hSL 100 57.0 28.5 32L4-PpLuc(GC)-Ndufb8-A64-C30-hSL 100 65.4 37.6 32L4-PpLuc(GC)-Cntn1004(V2)-A64-C30-hSL 100 71.0 47.7 32L4-PpLuc(GC)-A64-C30-hSL 100 45.2 21.87

[0545] Table 7 shows relative PpLuc expression normalized to RrLuc (mean values of three independent experiments are given).

8. Effect of Further 3UTRs on Protein Expression

[0546] To further investigate the effect of various 3UTRs on protein expression from mRNA, new mRNA constructs were prepared and those mRNAs containing different 3UTRs were compared to an mRNA lacking a 3UTR.

[0547] To this end, selected 3-UTR elements (gnas, morn2, ndufa1 and NDUFA1) were cloned into the vector PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 44, FIG. 10), which was constructed containing a T7 promoter and a GC-enriched sequence coding for Photinus pyralis luciferase (PpLuc(GC)). An A64 poly(A) sequence, followed by C30 and a histone stem-loop sequence, was inserted 3 of PpLuc(GC). The histone stem-loop sequence was followed by a restriction site used for linearization of the vector before in vitro transcription.

[0548] In particular, the following mRNAs were obtained from such vectors by in vitro transcription (the mRNA sequences are depicted in FIGS. 11 and 26 to 28:

PpLuc(GC)-gnas-A64-C30-hSL (SEQ ID NO. 45, FIG. 11);
PpLuc(GC)-morn2-A64-C30-hSL (SEQ ID NO. 384, FIG. 26);
PpLuc(GC)-ndufa1-A64-C30-hSL (SEQ ID NO. 385, FIG. 27); and

PpLuc(GC)-N DU FA1-A64-C30-hSL (SEQ ID NO. 386, FIG. 28).

[0549] 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 8 and FIG. 22).

TABLE-US-00016 TABLE 8 relative PpLuc expression normalized to RrLuc (mean values of 3 independent experiments are given). HeLa (expression in %) mRNA 24 h 48 h 72 h PpLuc(GC)-gnas-A64-C30-hSL 100 77.9 36.7 PpLuc(GC)-morn2-A64-C30-hSL 100 53.8 17.2 PpLuc(GC)-ndufa1-A64-C30-hSL 100 55.2 17.9 PpLuc(GC)-NDUFA1-A64-C30-hSL 100 66.9 29.4 PpLuc(GC)-A64-C30-hSL 100 41.5 9.6

[0550] These data and the data shown in FIG. 22 show that protein expression from mRNA containing 3-UTR elements according to the invention is prolonged.

9. Effect of Further 5UTRs on Protein Expression

[0551] To further investigate the effect of various 5UTRs on protein expression from mRNA, new mRNA constructs were prepared and those mRNAs containing different 5UTRs were compared to an mRNA lacking a 5UTR.

[0552] To this end, selected 5-UTR elements (mp68 and ndufa4) were cloned into the vector PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 44, FIG. 10), which was constructed containing a T7 promoter and a GC-enriched sequence coding for Photinus pyralis luciferase (PpLuc(GC)). An A64 poly(A) sequence, followed by C30 and a histone stem-loop sequence, was inserted 3 of PpLuc(GC). The histone stem-loop sequence was followed by a restriction site used for linearization of the vector before in vitro transcription.

[0553] In particular, the following mRNAs were obtained from such vectors by in vitro transcription (the mRNA sequences are depicted in FIGS. 29 and 30: Mp68-PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 387, FIG. 29); and Ndufa4-PpLuc(GC)-A64-C30-hSL (SEQ ID NO. 388, FIG. 30).

[0554] Human HDF and 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. Total protein expression (area under the curve) was calculated. The levels of the control construct without 5 UTR was set to 1 (see following Table 9 and FIGS. 23 and 24).

TABLE-US-00017 TABLE 9 total PpLuc expression normalized to RrLuc (mean RLU values are given). mRNA AUC HDF AUC HeLa PpLuc(GC)-A64-C30-hSL 1.0 1.0 Mp68-PpLuc(GC)-A64-C30-hSL 3.9 2.3 Ndufa4-PpLuc(GC)-A64-C30-hSL 4.0 2.0

[0555] These data and the data shown in FIGS. 23 and 24 show that protein expression from mRNA containing 5-UTR elements according to the invention is increased.