NUCLEIC ACID COMPRISING OR CODING FOR A HISTONE STEM-LOOP AND A POLY(A) SEQUENCE OR A POLYADENYLATION SIGNAL FOR INCREASING THE EXPRESSION OF AN ENCODED TUMOUR ANTIGEN

Abstract

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein. It also discloses its use for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the treatment of cancer or tumour diseases. The present invention further describes a method for increasing the expression of a peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

Claims

1-27. (canceled)

28. A nucleic acid molecule comprising: (I) a DNA molecule coding for, from 5 to 3: a) a polypeptide coding region, encoding a tumour antigen; b) a poly(A) sequence or a polyadenylation signal, and c) at least one histone stem-loop that encodes a RNA that specifically binds to stem-loop binding protein (SLBP) without a histone downstream element (HDE); or (II) a RNA molecule comprising, from 5 to 3: a) a polypeptide coding region, encoding a tumour antigen; b) a poly(A) sequence, and c) at least one histone stem-loop that specifically binds to SLBP without a HDE.

29. The nucleic acid molecule according to claim 28, wherein the tumour antigen is selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1.

30. The nucleic acid molecule of claim 28, wherein the molecule does not comprise a sequence encoding a reporter protein, a marker, or a selection protein.

31. The nucleic acid molecule of claim 28, wherein the nucleic acid is an RNA.

32. The nucleic acid molecule of claim 28, wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides.

33. The nucleic acid molecule of claim 28, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA.

34. The nucleic acid molecule of claim 28, wherein at least one guanosine, uridine, adenosine, thymidine, or cytidine position of the nucleic acid molecule is substituted with an analogue of these nucleotides selected from 2-amino-6-chloropurineriboside-5-triphosphate, 2-aminoadenosine-5-triphosphate, 2-thiocytidine-5-triphosphate, 2-thiouridine-5-triphosphate, 4-thiouridine-5-triphosphate, 5-aminoallylcytidine-5-triphosphate, 5-aminoallyluridine-5-triphosphate, 5-bromocytidine-5-triphosphate, 5-bromouridine-5-triphosphate, 5-iodocytidine-5-triphosphate, 5-iodouridine-5-triphosphate, 5-methylcytidine-5-triphosphate, 5-methyluridine-5-triphosphate, 6-azacytidine-5-triphosphate, 6-azauridine-5-triphosphate, 6-chloropurineriboside-5-triphosphate, 7-deazaadenosine-5-triphosphate, 7-deazaguanosine-5-triphosphate, 8-azaadenosine-5-triphosphate, 8-azidoadenosine-5-triphosphate, benzimidazole-riboside-5-triphosphate, N1-methyladenosine-5-triphosphate, N1-methylguanosine-5-triphosphate, N6-methyladenosine-5-triphosphate, 06-methylguanosine-5-triphosphate, pseudouridine-5-triphosphate, or puromycin-5-triphosphate, and xanthosine-5-triphosphate.

35. The nucleic acid sequence molecule of claim 28, wherein the G/C content of the polypeptide coding region is increased compared with the G/C content of the coding region of a wild-type nucleic acid encoding the tumour antigen.

36. The nucleic acid molecule of claim 31, wherein the RNA comprises a 5 cap structure and a poly(A) sequence of about 25 to about 400 adenosine nucleotides.

37. The nucleic acid molecule of claim 28, wherein the nucleic acid molecule comprises a sequence of at least 10 consecutive cytidines.

38. The nucleic acid molecule of claim 28, wherein the nucleic acid molecule further comprises a stabilizing sequence from the alpha globin 3 UTR, positioned 3 relative to the polypeptide coding region of the nucleic acid molecule.

39. A pharmaceutical composition comprising a nucleic acid molecule of claim 28 and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, further comprising an adjuvant.

41. The pharmaceutical composition of claim 39, wherein the composition further comprises a cationic or polycationic compound in complex with the nucleic acid molecule.

42. The pharmaceutical composition of claim 39, wherein the composition further comprises a polycationic polypeptide in complex with the nucleic acid molecule.

Description

FIGURES

[0279] The following Figures are intended to illustrate the invention further and shall not be construed to limit the present invention thereto.

[0280] FIG. 1: shows the histone stem-loop consensus sequence generated from metazoan and protozoan stem loop sequences (as reported by D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 4001 histone stem-loop sequences from metazoa and protozoa were aligned and the quantity of the occurring nucleotides is indicated for every position in the stem-loop sequence. The generated consensus sequence representing all nucleotides present in the sequences analyzed is given using the single-letter nucleotide code. In addition to the consensus sequence, sequences are shown representing at least 99%, 95% and 90% of the nucleotides present in the sequences analyzed.

[0281] FIG. 2: shows the histone stem-loop consensus sequence generated from protozoan stem loop sequences (as reported by D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 131 histone stem-loop sequences from protozoa were aligned and the quantity of the occurring nucleotides is indicated for every position in the stem-loop sequence. The generated consensus sequence representing all nucleotides present in the sequences analyzed is given using the single-letter nucleotide code. In addition to the consensus sequence, sequences are shown representing at least 99%, 95% and 90% of the nucleotides present in the sequences analyzed.

[0282] FIG. 3: shows the histone stem-loop consensus sequence generated from metazoan stem loop sequences (as reported by D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 3870 histone stem-loop sequences from metazoa were aligned and the quantity of the occurring nucleotides is indicated for every position in the stem-loop sequence. The generated consensus sequence representing all nucleotides present in the sequences analyzed is given using the single-letter nucleotide code. In addition to the consensus sequence, sequences are shown representing at least 99%, 95% and 90% of the nucleotides present in the sequences analyzed.

[0283] FIG. 4: shows the histone stem-loop consensus sequence generated from vertebrate stem loop sequences (as reported by D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 1333 histone stem-loop sequences from vertebrates were aligned and the quantity of the occurring nucleotides is indicated for every position in the stem-loop sequence. The generated consensus sequence representing all nucleotides present in the sequences analyzed is given using the single-letter nucleotide code. In addition to the consensus sequence, sequences are shown representing at least 99%, 95% and 90% of the nucleotides present in the sequences analyzed.

[0284] FIG. 5: shows the histone stem-loop consensus sequence generated from human (Homo sapiens) stem loop sequences (as reported by D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308). 84 histone stem-loop sequences from humans were aligned and the quantity of the occurring nucleotides is indicated for every position in the stem-loop sequence. The generated consensus sequence representing all nucleotides present in the sequences analyzed is given using the single-letter nucleotide code. In addition to the consensus sequence, sequences are shown representing at least 99%, 95% and 90% of the nucleotides present in the sequences analyzed.

[0285] FIGS. 6 to 21: show mRNAs from in vitro transcription. [0286] Given are the designation and the sequence of mRNAs obtained by in vitro transcription. [0287] The following abbreviations are used: [0288] ppLuc (GC): GC-enriched mRNA sequence coding for Photinus pyralis luciferase [0289] ag: 3 untranslated region (UTR) of the alpha globin gene [0290] A64: poly(A)-sequence with 64 adenylates [0291] A120: poly(A)-sequence with 120 adenylates [0292] histoneSL: histone stem-loop [0293] aCPSL: stem loop which has been selected from a library for its specific binding of the ?CP-2KL protein [0294] PolioCL: 5 clover leaf from Polio virus genomic RNA [0295] G30: poly(G) sequence with 30 guanylates [0296] U30: poly(U) sequence with 30 uridylates [0297] SL: unspecific/artificial stem-loop [0298] N32: unspecific sequence of 32 nucleotides [0299] NY-ESO-1 (G/C): GC-enriched mRNA sequence coding for the human tumour antigen NY-ESO-1 [0300] Survivin(G/C): GC-enriched mRNA sequence coding for the human tumour antigen Survivin [0301] MAGE-C1(G/C): GC-enriched mRNA sequence coding for the human tumour antigen MAGE-C1 [0302] Within the sequences, the following elements are highlighted: coding region (ORF) (capital letters), ag (bold), histoneSL (underlined), further distinct sequences tested (italic).

[0303] FIG. 6: shows the mRNA sequence of ppLuc(GC)-ag (SEQ ID NO: 43). [0304] By linearization of the original vector at the restriction site immediately following the alpha-globin 3-UTR (ag), mRNA is obtained lacking a poly(A) sequence.

[0305] FIG. 7: shows the mRNA sequence of ppLuc(GC)-ag-A64 (SEQ ID NO: 44). [0306] By linearization of the original vector at the restriction site immediately following the A64 poly(A)-sequence, mRNA is obtained ending with an A64 poly(A) sequence.

[0307] FIG. 8: shows the mRNA sequence of ppLuc(GC)-ag-histoneSL (SEQ ID NO: 45). [0308] The A64 poly(A) sequence was replaced by a histoneSL. The histone stem-loop sequence used in the examples was obtained from Cakmakci et al. (2008). Molecular and Cellular Biology, 28(3), 1182-1194.

[0309] FIG. 9: shows the mRNA sequence of ppLuc(GC)-ag-A64-histoneSL (SEQ ID NO: 46). [0310] The histoneSL was appended 3 of A64 poly(A).

[0311] FIG. 10: shows the mRNA sequence of ppLuc(GC)-ag-A120 (SEQ ID NO: 47). [0312] The A64 poly(A) sequence was replaced by an A120 poly(A) sequence.

[0313] FIG. 11: shows the mRNA sequence of ppLuc(GC)-ag-A64-ag (SEQ ID NO: 48). A second alpha-globin 3-UTR was appended 3 of A64 poly(A).

[0314] FIG. 12: shows the mRNA sequence of ppLuc(GC)-ag-A64-aCPSL (SEQ ID NO: 49). [0315] A stem loop was appended 3 of A64 poly(A). The stem loop has been selected from a library for its specific binding of the ?CP-2KL protein (Thisted et al., (2001), The Journal of Biological Chemistry, 276(20), 17484-17496). ?CP-2KL is an isoform of ?CP-2, the most strongly expressed ?CP protein (alpha-globin mRNA poly(C) binding protein) (Makeyev et al., (2000), Genomics, 67(3), 301-316), a group of RNA binding proteins, which bind to the alpha-globin 3-UTR (Chkheidze et al., (1999), Molecular and Cellular Biology, 19(7), 4572-4581).

[0316] FIG. 13: shows the mRNA sequence of ppLuc(GC)-ag-A64-PolioCL (SEQ ID NO: 50). [0317] The 5 clover leaf from Polio virus genomic RNA was appended 3 of A64 poly(A).

[0318] FIG. 14: shows the mRNA sequence of ppLuc(GC)-ag-A64-G30 (SEQ ID NO: 51) [0319] A stretch of 30 guanylates was appended 3 of A64 poly(A).

[0320] FIG. 15: shows the mRNA sequence of ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 52) [0321] A stretch of 30 uridylates was appended 3 of A64 poly(A).

[0322] FIG. 16: shows the mRNA sequence of ppLuc(GC)-ag-A64-SL (SEQ ID NO: 53) [0323] A stem loop was appended 3 of A64 poly(A). The upper part of the stem and the loop were taken from (Babendure et al., (2006), RNA (New York, N.Y.), 12(5), 851-861). The stem loop consists of a 17 base pair long, CG-rich stem and a 6 base long loop.

[0324] FIG. 17: shows ppLuc(GC)-ag-A64-N32 (SEQ ID NO: 54) [0325] By linearization of the original vector at an alternative restriction site, mRNA is obtained with 32 additional nucleotides following poly(A).

[0326] FIG. 18: shows the mRNA sequence of NY-ESO-1(GC)-ag-A64-C30 (SEQ ID NO: 55)

[0327] FIG. 19: shows the mRNA sequence of NY-ESO-1(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 56)

[0328] FIG. 20: shows the mRNA sequence of Survivin(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 57)

[0329] FIG. 21: shows the mRNA sequence of MAGE-C1(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 58)

[0330] FIG. 22: shows that the combination of poly(A) and histoneSL increases protein expression from mRNA in a synergistic manner. [0331] The effect of poly(A) sequence, histoneSL, and the combination of poly(A) and histoneSL on luciferase expression from mRNA was examined. Therefore different mRNAs were electroporated into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after transfection. Little luciferase is expressed from mRNA having neither poly(A) sequence nor histoneSL. Both a poly(A) sequence or the histoneSL increase the luciferase level. Strikingly however, the combination of poly(A) and histoneSL further strongly increases the luciferase level, manifold above the level observed with either of the individual elements, thus acting synergistically. Data are graphed as mean RLU?SD (relative light units ?standard deviation) for triplicate transfections. Specific RLU are summarized in Example 14.2.

[0332] FIG. 23: shows that the combination of poly(A) and histoneSL increases protein expression from mRNA irrespective of their order. [0333] The effect of poly(A) sequence, histoneSL, the combination of poly(A) and histoneSL, and their order on luciferase expression from mRNA was examined. Therefore different mRNAs were lipofected into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after the start of transfection. Both an A64 poly(A) sequence or the histoneSL give rise to comparable luciferase levels. Increasing the length of the poly(A) sequence from A64 to A120 or to A300 increases the luciferase level moderately. In contrast, the combination of poly(A) and histoneSL increases the luciferase level much further than lengthening of the poly(A) sequence. The combination of poly(A) and histoneSL acts synergistically as it increases the luciferase level manifold above the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and histoneSL is seen irrespective of the order of poly(A) and histoneSL and irrespective of the length of poly(A) with A64-histoneSL or histoneSL-A250 mRNA. Data are graphed as mean RLU?SD for triplicate transfections. Specific RLU are summarized in Example 14.3.

[0334] FIG. 24: shows that the rise in protein expression by the combination of poly(A) and histoneSL is specific. [0335] The effect of combining poly(A) and histoneSL or poly(A) and alternative sequences on luciferase expression from mRNA was examined. Therefore different mRNAs were electroporated into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after transfection. Both a poly(A) sequence or the histoneSL give rise to comparable luciferase levels. The combination of poly(A) and histoneSL strongly increases the luciferase level, manifold above the level observed with either of the individual elements, thus acting synergistically. In contrast, combining poly(A) with any of the other sequences is without effect on the luciferase level compared to mRNA containing only a poly(A) sequence. Thus, the combination of poly(A) and histoneSL acts specifically and synergistically. Data are graphed as mean RLU?SD for triplicate transfections. Specific RLU are summarized in Example 14.4.

[0336] FIG. 25: shows that the combination of poly(A) and histoneSL increases protein expression from mRNA in a synergistic manner in vivo. [0337] The effect of poly(A) sequence, histoneSL, and the combination of poly(A) and histoneSL on luciferase expression from mRNA in vivo was examined. Therefore different mRNAs were injected intradermally into mice. Mice were sacrificed 16 hours after injection and Luciferase levels at the injection sites were measured. Luciferase is expressed from mRNA having either a histoneSL or a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL strongly increases the luciferase level, manifold above the level observed with either of the individual elements, thus acting synergistically. Data are graphed as mean RLU?SEM (relative light units ?standard error of mean). Specific RLU are summarized in Example 14.5.

[0338] FIG. 26: shows that the combination of poly(A) and histoneSL increases NY-ESO-1 protein expression from mRNA. [0339] The effect of poly(A) sequence and the combination of poly(A) and histoneSL on NY-ESO-1 expression from mRNA was examined. Therefore different mRNAs were electroporated into HeLa cells. NY-ESO-1 levels were measured at 24 hours after transfection by flow cytometry. NY-ESO-1 is expressed from mRNA having only a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL strongly increases the NY-ESO-1 level, manifold above the level observed with only a poly(A) sequence. Data are graphed as counts against fluorescence intensity. Median fluorescence intensities (MFI) are summarized in Example 14.6.

[0340] FIG. 27: shows that the combination of poly(A) and histoneSL increases the level of antibodies elicited by vaccination with mRNA. [0341] The effect of poly(A) sequence and the combination of poly(A) and histoneSL on the induction of anti NY-ESO-1 antibodies elicited by vaccination with mRNA was examined. Therefore C57BL/6 mice were vaccinated intradermally with different mRNAs complexed with protamine. The level of NY-ESO-1-specific antibodies in vaccinated and control mice was analyzed by ELISA with serial dilutions of sera. Anti NY-ESO-1 IgG2a[b] is induced by mRNA having only a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL strongly increases the anti NY-ESO-1 IgG2a[b] level, manifold above the level observed with only a poly(A) sequence. Data are graphed as mean endpoint titers. Mean endpoint titers are summarized in Example 14.7.

EXAMPLES

[0342] The following Examples are intended to illustrate the invention further and shall not be construed to limit the present invention thereto.

[0343] 1. Generation of Histone-Stem-Loop Consensus Sequences

[0344] Prior to the experiments, histone stem-loop consensus sequences were determined on the basis of metazoan and protozoan histone stem-loop sequences. Sequences were taken from the supplement provided by Lopez et al. (D?vila L?pez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10. doi:10.1261/rna.782308), who identified a large number of natural histone stem-loop sequences by searching genomic sequences and expressed sequence tags. First, all sequences from metazoa and protozoa (4001 sequences), or all sequences from protozoa (131 sequences) or alternatively from metazoa (3870 sequences), or from vertebrates (1333 sequences) or from humans (84 sequences) were grouped and aligned. Then, the quantity of the occurring nucleotides was determined for every position. Based on the tables thus obtained, consensus sequences for the 5 different groups of sequences were generated representing all nucleotides present in the sequences analyzed. In addition, more restrictive consensus sequences were also obtained, increasingly emphasizing conserved nucleotides

[0345] 2. Preparation of DNA-Templates

[0346] A vector for in vitro transcription was constructed containing a T7 promoter followed by a GC-enriched sequence coding for Photinus pyralis luciferase (ppLuc(GC)), the center part of the 3 untranslated region (UTR) of alpha-globin (ag), and a poly(A) sequence. The poly(A) sequence was immediately followed by a restriction site used for linearization of the vector before in vitro transcription in order to obtain mRNA ending in an A64 poly(A) sequence. mRNA obtained from this vector accordingly by in vitro transcription is designated as ppLuc(GC)-ag-A64.

[0347] Linearization of this vector at alternative restriction sites before in vitro transcription allowed to obtain mRNA either extended by additional nucleotides 3 of A64 or lacking A64. In addition, the original vector was modified to include alternative sequences. In summary, the following mRNAs were obtained from these vectors by in vitro transcription (mRNA sequences are given in FIGS. 6 to 17):

TABLE-US-00017 (SEQ ID NO: 43) ppLuc(GC)-ag (SEQ ID NO: 44) ppLuc(GC)-ag-A64 (SEQ ID NO: 45) ppLuc(GC)-ag-histoneSL (SEQ ID NO: 46) ppLuc(GC)-ag-A64-histoneSL (SEQ ID NO: 47) ppLuc(GC)-ag-A120 (SEQ ID NO: 48) ppLuc(GC)-ag-A64-ag (SEQ ID NO: 49) ppLuc(GC)-ag-A64-aCPSL (SEQ ID NO: 50) ppLuc(GC)-ag-A64-PolioCL (SEQ ID NO: 51) ppLuc(GC)-ag-A64-G30 (SEQ ID NO: 52) ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 53) ppLuc(GC)-ag-A64-SL (SEQ ID NO: 54) ppLuc(GC)-ag-A64-N32

[0348] Furthermore DNA plasmid sequences coding for the tumour antigens NY-ESO-1, Survivin and MAGE-C1 were prepared accordingly as described above.

[0349] In summary, the following mRNAs were obtained from these vectors by in vitro transcription (mRNA sequences are given in FIGS. 18 to 21):

TABLE-US-00018 (SEQ ID NO: 55) NY-ESO-1(GC)-ag-A62-C30 (SEQ ID NO: 56) NY-ESO-1(GC)-ag-A62-C30-histoneSL (SEQ ID NO: 57) Survivin(GC)-ag-A62-C30-histoneSL (SEQ ID NO: 58) MAGE-C1(GC)-ag-A64-C30-histoneSL

[0350] 3. In Vitro Transcription

[0351] The DNA-template according to Example 2 was linearized and transcribed in vitro using T7-Polymerase. The DNA-template was then digested by DNase-treatment. All 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.

[0352] 4. Enzymatic Adenylation of mRNA

[0353] Two mRNAs were enzymatically adenylated: [0354] ppLuc(GC)-ag-A64 and ppLuc(GC)-ag-histoneSL.

[0355] To this end, RNA was incubated with E. coli Poly(A)-polymerase and ATP (Poly(A) Polymerase Tailing Kit, Epicentre, Madison, USA) following the manufacturer's instructions. mRNA with extended poly(A) sequence was purified and resuspended in water. The length of the poly(A) sequence was determined via agarose gel electrophoresis. Starting mRNAs were extended by approximately 250 adenylates, the mRNAs obtained are designated as ppLuc(GC)-ag-A300 and ppLuc(GC)-ag-histoneSL-A250, respectively.

[0356] 5. Luciferase Expression by mRNA Electroporation

[0357] HeLa cells were trypsinized and washed in opti-MEM. 1?10.sup.5 cells in 200 ?l of opti-MEM each were electroporated with 0.5 ?g of ppLuc-encoding mRNA. As a control, mRNA not coding for ppLuc was electroporated separately. Electroporated cells were seeded in 24-well plates in 1 ml of RPMI 1640 medium. 6, 24, or 48 hours after transfection, medium was aspirated and cells were lysed in 200 ?l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). Lysates were stored at ?20? C. until ppLuc activity was measured.

[0358] 6. Luciferase Expression by mRNA Lipofection

[0359] HeLa cells were seeded in 96 well plates at a density of 2?10.sup.4 cells per well. The following day, cells were washed in opti-MEM and then transfected with 0.25 ?g of Lipofectin-complexed ppLuc-encoding mRNA in 150 ?l of opti-MEM. As a control, mRNA not coding for ppLuc was lipofected separately. In some wells, opti-MEM was aspirated and cells were lysed in 200 ?l of lysis buffer 6 hours after the start of transfection. In the remaining wells, opti-MEM was exchanged for RPMI 1640 medium at that time. In these wells, medium was aspirated and cells were lysed in 200 ?l of lysis buffer 24 or 48 hours after the start of transfection. Lysates were stored at ?20? C. until ppLuc activity was measured.

[0360] 7. Luciferase Measurement [0361] ppLuc activity was measured as relative light units (RLU) in a BioTek SynergyHT plate reader at 5 seconds measuring time using 50 ?l of lysate and 200 ?l of luciferin buffer (25 mM Glycylglycin, pH 7.8 (NaOH), 15 mM MgSO.sub.4, 2 mM ATP, 75 ?M luciferin). Specific RLU were calculated by subtracting RLU of the control RNA from total RLU.

[0362] 8. Luciferase Expression by Intradermal mRNA Injection (Luciferase Expression In Vivo)

[0363] Mice were anaesthetized with a mixture of Rompun and Ketavet. Each ppLuc-encoding mRNA was injected intradermally (0.5 ?g of mRNA in 50 ?l per injection). As a control, mRNA not coding for ppLuc was injected separately. 16 hours after injection, mice were sacrificed and tissue collected. Tissue samples were flash frozen in liquid nitrogen and lysed in a tissue lyser (Qiagen) in 800 ?l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). Subsequently samples were centrifuged at 13500 rpm at 4? C. for 10 minutes. Lysates were stored at ?80? C. until ppLuc activity was measured (see 7. luciferase measurement).

[0364] 9. NY-ESO-1 Expression by mRNA Electroporation

[0365] HeLa cells were trypsinized and washed in opti-MEM. 2?10.sup.5 cells in 200 ?l of opti-MEM were electroporated with 10 ?g of NY-ESO-1-encoding mRNA. Cells from three electroporations were combined and seeded in a 6-well plate in 2 ml of RPMI 1640 medium. 24 hours after transfection, cells were harvested and transferred into a 96 well V-bottom plate (2 wells per mRNA). Cells were washed with phosphate buffered saline (PBS) and permeabilized with 200 ?l per well of Cytofix/Cytoperm (Becton Dickinson (BD)). After 15 minutes, cells were washed with PERM/WASH? buffer (BD). Then, cells were incubated for 1 hour at room temperature with either mouse anti-NY-ESO-1 IgG1 or an isotype control (20 ?g/ml). Cells were washed twice with PERM/WASH? buffer again. Next, cells were incubated for 1 hour at 4? C. with a 1:500 dilution of Alexa-647 coupled goat-anti-mouse IgG. Finally, cells were washed twice with PERM/WASH? buffer. Cells were resuspended in 200 ?l of buffer (PBS, 2% FCS, 2 mM EDTA, 0.01% sodium azide). NY-ESO-1 expression was quantified by flow cytometry as median fluorescence intensity (MFI).

[0366] 10. Induction of Anti NY-ESO-1 Antibodies by Vaccination with mRNA

[0367] C57BL/6 mice were vaccinated intradermally with NY-ESO-1-encoding mRNA complexed with protamine (5 times in 14 days). Control mice were treated with buffer. The level of NY-ESO-1-specific antibodies in vaccinated and control mice was analyzed 8 days after the last vaccination by ELISA: 96 well ELISA plates (Nunc) were coated with 100 ?l per well of 10 ?g/ml recombinant NY-ESO-1 protein for 16 hours at 4? C. Plates were washed two times with wash buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent). To block unspecific binding, plates were then incubated for 2 hours at 37? C. with blocking buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent, 1% BSA). After blocking, 100 ?l per well of serially diluted mouse sera were added and incubated for 4 hours at room temperature. Plates were then washed three times with wash buffer. Next, 100 ?l per well of biotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences) diluted 1:600 in blocking buffer was allowed to bind for 1 hour at room temperature. Plates were washed again three times with wash buffer, followed by incubation for 30 minutes at room temperature with 100 ?l per well of horseradish peroxidase-coupled streptavidin. After four washes with wash buffer, 100 ?l per well of 3,3,5,5-tetramethylbenzidine (Thermo Scientific) was added. Upon the resulting change in color 100 ?l per well of 20% sulfuric acid was added. Absorbance was measured at 405 nm.

[0368] 11. Induction of Anti Survivin Antibodies by Vaccination with mRNA

[0369] C57BL/6 mice were vaccinated intradermally with Survivin-encoding mRNA complexed with protamine (5 times in 14 days). Control mice were treated with buffer. The level of Survivin-specific antibodies in vaccinated and control mice was analyzed 8 days after the last vaccination by ELISA: 96 well ELISA plates (Nunc) were coated with 100 ?l per well of 10 ?g/ml recombinant Survivin protein for 16 hours at 4? C. Plates were washed two times with wash buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent). To block unspecific binding, plates were then incubated for 2 hours at 37? C. with blocking buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent, 1% BSA). After blocking, 100 ?l per well of serially diluted mouse sera were added and incubated for 4 hours at room temperature. Plates were then washed three times with wash buffer. Next, 100 ?l per well of biotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences) diluted 1:600 in blocking buffer was allowed to bind for 1 hour at room temperature. Plates were washed again three times with wash buffer, followed by incubation for 30 minutes at room temperature with 100 ?l per well of horseradish peroxidase-coupled streptavidin. After four washes with wash buffer, 100 ?l per well of 3,3,5,5-tetramethylbenzidine (Thermo Scientific) was added. Upon the resulting change in color 100 ?l per well of 20% sulfuric acid was added. Absorbance was measured at 405 nm.

[0370] 12. Induction of Anti MAGE-C1 Antibodies by Vaccination with mRNA

[0371] C57BL/6 mice were vaccinated intradermally with MAGE-C1-encoding mRNA complexed with protamine (5 times in 14 days). Control mice were treated with buffer. The level of MAGE-C1-specific antibodies in vaccinated and control mice was analyzed 8 days after the last vaccination by ELISA: 96 well ELISA plates (Nunc) were coated with 100 ?l per well of 10 ?g/ml recombinant MAGE-C1 protein for 16 hours at 4? C. Plates were washed two times with wash buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent). To block unspecific binding, plates were then incubated for 2 hours at 37? C. with blocking buffer (PBS, 0.05% TWEEN? 20 non-ionic detergent, 1% BSA). After blocking, 100 ?l per well of serially diluted mouse sera were added and incubated for 4 hours at room temperature. Plates were then washed three times with wash buffer. Next, 100 ?l per well of biotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences) diluted 1:600 in blocking buffer was allowed to bind for 1 hour at room temperature. Plates were washed again three times with wash buffer, followed by incubation for 30 minutes at room temperature with 100 ?l per well of horseradish peroxidase-coupled streptavidin. After four washes with wash buffer, 100 ?l per well of 3,3,5,5-tetramethylbenzidine (Thermo Scientific) was added. Upon the resulting change in color 100 ?l per well of 20% sulfuric acid was added. Absorbance was measured at 405 nm.

[0372] 13. Detection of an Antigen-Specific Cellular Immune Response (T Cell Immune Response) by ELISPOT:

[0373] C57BL/6 mice are vaccinated intradermally with MAGE-C1 encoding mRNA complexed with protamine (5 times in 14 days). Control mice are treated with buffer. 1 week after the last vaccination mice are sacrificed, the spleens are removed and the splenocytes are isolated. The splenocytes are restimulated for 7 days in the presence of peptides from the above antigen (peptide library) or coincubated with dendritic cells generated from bone marrow cells of native syngeneic mice, which are electroporated with mRNA coding for the antigen. To determine an antigen-specific cellular immune response INFgamma secretion was measured after re-stimulation. For detection of INFgamma a coat multiscreen plate (Millipore) is incubated overnight with coating buffer 0.1 M carbonate-bicarbonate buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF? (BD Pharmingen, Heidelberg, Germany). Stimulators and effector cells are incubated together in the plate in the ratio of 1:20 for 24 h. The plate is washed with 1?PBS and incubated with a biotin-coupled secondary antibody. After washing with 1?PBS/0.05% TWEEN? 20 non-ionic detergent, the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) is added to the plate and the conversion of the substrate could be detected visually.

[0374] 14. Results

[0375] 14.1 Histone Stem-Loop Sequences:

[0376] In order to characterize histone stem-loop sequences, sequences from metazoa and protozoa (4001 sequences), or from protozoa (131 sequences) or alternatively from metazoa (3870 sequences), or from vertebrates (1333 sequences) or from humans (84 sequences) were grouped and aligned. Then, the quantity of the occurring nucleotides was determined for every position. Based on the tables thus obtained, consensus sequences for the 5 different groups of sequences were generated representing all nucleotides present in the sequences analyzed. Within the consensus sequence of metazoa and protozoa combined, 3 nucleotides are conserved, a T/U in the loop and a G and a C in the stem, forming a base pair. Structurally, typically a 6 base-pair stem and a loop of 4 nucleotides is formed. However, deviating structures are common: Of 84 human histone stem-loops, two contain a stem of only 5 nucleotides comprising 4 base-pairs and one mismatch. Another human histone stem-loop contains a stem of only 5 base-pairs. Four more human histone stem-loops contain a 6 nucleotide long stem, but include one mismatch at three different positions, respectively. Furthermore, four human histone stem-loops contain one wobble base-pair at two different positions, respectively. Concerning the loop, a length of 4 nucleotides seems not to be strictly required, as a loop of 5 nucleotides has been identified in D. discoideum.

[0377] In addition to the consensus sequences representing all nucleotides present in the sequences analyzed, more restrictive consensus sequences were also obtained, increasingly emphasizing conserved nucleotides. In summary, the following sequences were obtained: [0378] (Cons): represents all nucleotides present [0379] (99%): represents at least 99% of all nucleotides present [0380] (95%): represents at least 95% of all nucleotides present [0381] (90%): represents at least 90% of all nucleotides present

[0382] The results of the analysis of histone stem-loop sequences are summarized in the following Tables 1 to 5 (see also FIG. 1 to 5):

TABLE-US-00019 TABLE 1 Metazoan and protozoan histone stem-loop consensus sequence: (based on an alignment of 4001 metazoan and protozoan histone stem-loop sequences) (see also FIG. 1) < < < < < < .Math. .Math. # A 2224 1586 3075 2872 1284 184 0 13 12 9 1 47 59 # T 172 188 47 205 19 6 0 569 1620 199 3947 3830 3704 # C 1557 2211 875 918 2675 270 0 3394 2342 3783 51 119 227 # G 25 16 4 6 23 3541 4001 25 27 10 2 5 11 Cons N* N* N N N N G N N N N N N 99% H* H* H H V V G Y Y Y Y H H 95% M* H* M H M S G Y Y Y T T Y 90% M* M* M M M S G Y Y C T T T .Math. .Math. > > > > > > # A 0 675 3818 195 1596 523 0 14 3727 61 771 2012 2499 # T 4001 182 1 21 15 11 0 179 8 64 557 201 690 # C 0 3140 7 50 31 16 4001 3543 154 3870 2636 1744 674 # G 0 4 175 3735 2359 3451 0 265 112 4 37 43 138 Cons T N N N N N C N N N N* N* N* 99% T H R V V R C B V H H* N* N* 95% T M A R R R C S M C H* H* H* 90% T M A G R R C S A C H* M* H*

TABLE-US-00020 TABLE 2 Protozoan histone stem-loop consensus sequence: (based on an alignment of 131 protozoan histone stem-loop sequences) (see also FIG. 2) < < < < < < .Math. .Math. .Math. .Math. > > > > > > # A 52 32 71 82 76 13 0 12 12 9 1 46 3 0 75 82 53 79 20 0 4 94 17 35 74 56 # T 20 32 37 21 8 3 0 21 85 58 86 70 65 131 28 1 17 13 10 0 15 7 31 32 20 28 # C 45 59 20 25 38 0 0 86 8 54 42 13 58 0 27 2 6 31 10 131 112 5 82 58 30 40 # G 14 8 3 3 9 115 131 12 26 10 2 2 5 0 1 46 55 8 91 0 0 25 1 6 7 7 Cons N* N* N N N D G N N N N N N T N N N N N C H N N N* N* N* 99% N* N* N N N D G N N N B N N T H V N N N C H N H N* N* N* 95% N* N* H H N R G N N N Y H B T H R D N N C Y D H H* N* N* 90% N* H* H H V R G N D B Y H Y T H R D H N C Y R H H* H* H*

TABLE-US-00021 TABLE 3 Metazoan histone stem-loop consensus sequence: (based on an alignment of 3870 (including 1333 vertebrate sequences) metazoan histone stem-loop sequences) (see also FIG. 3) < < < < < < .Math. .Math. # A 2172 1554 3004 2790 1208 171 0 1 0 0 0 1 56 # T 152 156 10 184 11 3 0 548 1535 141 3861 3760 3639 # C 1512 2152 855 893 2637 270 0 3308 2334 3729 9 106 169 # G 11 8 1 3 14 3426 3870 13 1 0 0 3 6 Cons N* N* N N N N G N B Y Y N N 99% H* H* M H M V G Y Y Y T Y H 95% M* M* M M M S G Y Y C T T Y 90% M* M* M M M S G Y Y C T T T .Math. .Math. > > > > > > # A 0 600 3736 142 1517 503 0 10 3633 44 736 1938 2443 # T 3870 154 0 4 2 1 0 164 1 33 525 181 662 # C 0 3113 5 44 0 6 3870 3431 149 3788 2578 1714 634 # G 0 3 129 3680 2351 3360 0 265 87 3 31 36 131 Cons T N V N D N C N N N N* N* N* 99% T H R V R R C B V M H* H* N* 95% T M A G R R C S M C H* H* H* 90% T M A G R R C S A C H* M* H*

TABLE-US-00022 TABLE 4 Vertebrate histone stem-loop consensus sequence: (based on an alignment of 1333 vertebrate histone stem-loop sequences) (see also FIG. 4) < < < < < < .Math. .Math. # A 661 146 1315 1323 920 8 0 1 0 0 0 1 4 # T 63 121 2 2 6 2 0 39 1217 2 1331 1329 1207 # C 601 1062 16 6 403 1 0 1293 116 1331 2 0 121 # G 8 4 0 2 4 1322 1333 0 0 0 0 3 1 Cons N* N* H N N N G H Y Y Y D N 99% H* H* M A M G G Y Y C T T Y 95% H* H* A A M G G C Y C T T Y 90% M* M* A A M G G C T C T T T .Math. .Math. > > > > > > # A 0 441 1333 0 1199 21 0 1 1126 26 81 380 960 # T 1333 30 0 1 0 1 0 2 1 22 91 91 12 # C 0 862 0 2 0 0 1333 1328 128 1284 1143 834 361 # G 0 0 0 1330 134 1311 0 2 78 1 18 28 0 Cons T H A B R D C N N N N* N* H* 99% T H A G R R C C V H N* N* M* 95% T M A G R G C C V C H* H* M* 90% T M A G R G C C M C Y* M* M*

TABLE-US-00023 TABLE 5 Homo sapiens histone stem-loop consensus sequence: (based on an alignment of 84 human histone stem-loop sequences) (see also FIG. 5) < < < < < < .Math. .Math. .Math. .Math. > > > > > > # A 10 17 84 84 76 1 0 1 0 0 0 1 0 0 12 84 0 65 3 0 0 69 5 0 10 64 # T 8 6 0 0 2 2 0 1 67 0 84 80 81 84 5 0 0 0 0 0 0 0 4 25 24 3 # C 62 61 0 0 6 0 0 82 17 84 0 0 3 0 67 0 1 0 0 84 84 5 75 57 44 17 # G 4 0 0 0 0 81 84 0 0 0 0 3 0 0 0 0 83 19 81 0 0 10 0 2 6 0 Cons N* H* A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 99% N* H* A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 95% H* H* A A M G G C Y C T T T T H A G R G C C V M Y* N* M* 90% H* M* A A A G G C Y C T T T T M A G R G C C R M Y* H* M*

[0383] Wherein the used abbreviations were defined as followed:

TABLE-US-00024 abbreviation Nucleotide bases remark G G Guanine A A Adenine T T Thymine U U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or C Amino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2H bonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/U D G or A or T/U Not C N* G or C or T/U or A Any base present or not Base may be present or not

[0384] 14.2 the Combination of Poly(A) and histoneSL Increases Protein Expression from mRNA in a Synergistic Manner.

[0385] To investigate the effect of the combination of poly(A) and histoneSL on protein expression from mRNA, mRNAs with different sequences 3 of the alpha-globin 3-UTR were synthesized: mRNAs either ended just 3 of the 3-UTR, thus lacking both poly(A) sequence and histoneSL, or contained either an A64 poly(A) sequence or a histoneSL instead, or both A64 poly(A) and histoneSL 3 of the 3-UTR. Luciferase-encoding mRNAs or control mRNA were electroporated into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after transfection (see following Table 6 and FIG. 22).

TABLE-US-00025 TABLE 6 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hours ppLuc(GC)-ag-A64-histoneSL 466553 375169 70735 ppLuc(GC)-ag-histoneSL 50947 3022 84 ppLuc(GC)-ag-A64 10471 19529 4364 ppLuc(GC)-ag 997 217 42

[0386] Little luciferase was expressed from mRNA having neither poly(A) sequence nor histoneSL. Both a poly(A) sequence or the histoneSL increased the luciferase level to a similar extent. Either mRNA gave rise to a luciferase level much higher than did mRNA lacking both poly(A) and histoneSL. Strikingly however, the combination of poly(A) and histoneSL further strongly increased the luciferase level, manifold above the level observed with either of the individual elements. The magnitude of the rise in luciferase level due to combining poly(A) and histoneSL in the same mRNA demonstrates that they are acting synergistically.

[0387] The synergy between poly(A) and histoneSL was quantified by dividing the signal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals from histoneSL mRNA (?/+) plus poly(A) mRNA (+/?) (see following Table 7).

TABLE-US-00026 TABLE 7 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + + 466553 375169 70735 ? + 50947 3022 84 + ? 10471 19529 4364 Synergy 7.6 16.6 15.9

[0388] The factor thus calculated specifies how much higher the luciferase level from mRNA combining poly(A) and histoneSL is than would be expected if the effects of poly(A) and histoneSL were purely additive. The luciferase level from mRNA combining poly(A) and histoneSL was up to 16.6 times higher than if their effects were purely additive. This result confirms that the combination of poly(A) and histoneSL effects a markedly synergistic increase in protein expression.

[0389] 14.3 the Combination of Poly(A) and histoneSL Increases Protein Expression from mRNA Irrespective of their Order.

[0390] The effect of the combination of poly(A) and histoneSL might depend on the length of the poly(A) sequence and the order of poly(A) and histoneSL. Thus, mRNAs with increasing poly(A) sequence length and mRNA with poly(A) and histoneSL in reversed order were synthesized: Two mRNAs contained 3 of the 3-UTR either an A120 or an A300 poly(A) sequence. One further mRNA contained 3 of the 3-UTR first a histoneSL followed by an A250 poly(A) sequence. Luciferase-encoding mRNAs or control mRNA were lipofected into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after the start of transfection (see following Table 8 and FIG. 23).

TABLE-US-00027 TABLE 8 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hours ppLuc(GC)-ag-histoneSL-A250 98472 734222 146479 ppLuc(GC)-ag-A64-histoneSL 123674 317343 89579 ppLuc(GC)-ag-histoneSL 7291 4565 916 ppLuc(GC)-ag-A300 4357 38560 11829 ppLuc(GC)-ag-A120 4371 45929 10142 ppLuc(GC)-ag-A64 1928 26781 537

[0391] Both an A64 poly(A) sequence or the histoneSL gave rise to comparable luciferase levels. In agreement with the previous experiment did the combination of A64 and histoneSL strongly increase the luciferase level, manifold above the level observed with either of the individual elements. The magnitude of the rise in luciferase level due to combining poly(A) and histoneSL in the same mRNA demonstrates that they are acting synergistically. The synergy between A64 and histoneSL was quantified as before based on the luciferase levels of A64-histoneSL, A64, and histoneSL mRNA (see following Table 9). The luciferase level from mRNA combining A64 and histoneSL was up to 61.7 times higher than if the effects of poly(A) and histoneSL were purely additive.

TABLE-US-00028 TABLE 9 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + + 123674 317343 89579 ? + 7291 4565 916 + ? 1928 26781 537 Synergy 13.4 10.1 61.7

[0392] In contrast, increasing the length of the poly(A) sequence from A64 to A120 or to A300 increased the luciferase level only moderately (see Table 8 and FIG. 19). mRNA with the longest poly(A) sequence, A300, was also compared to mRNA in which a poly(A) sequence of similar length was combined with the histoneSL, histoneSL-A250. In addition to having a long poly(A) sequence, the order of histoneSL and poly(A) is reversed in this mRNA relative to A64-histoneSL mRNA. The combination of A250 and histoneSL strongly increased the luciferase level, manifold above the level observed with either histoneSL or A300. Again, the synergy between A250 and histoneSL was quantified as before comparing RLU from histoneSL-A250 mRNA to RLU from A300 mRNA plus histoneSL mRNA (see following Table 10). The luciferase level from mRNA combining A250 and histoneSL was up to 17.0 times higher than if the effects of poly(A) and histoneSL were purely additive.

TABLE-US-00029 TABLE 10 RLU at RLU at RLU at histoneSL A250/A300 6 hours 24 hours 48 hours + + 98472 734222 146479 + ? 7291 4565 916 ? + 4357 38560 11829 Synergy 8.5 17.0 11.5

[0393] In summary, a highly synergistic effect of the combination of histoneSL and poly(A) on protein expression from mRNA has been demonstrated for substantially different lengths of poly(A) and irrespective of the order of poly(A) and histoneSL.

[0394] 14.4 the Rise in Protein Expression by the Combination of Poly(A) and histoneSL is Specific

[0395] To investigate whether the effect of the combination of poly(A) and histoneSL on protein expression from mRNA is specific, mRNAs with alternative sequences in combination with poly(A) were synthesized: These mRNAs contained 3 of A64 one of seven distinct sequences, respectively. Luciferase-encoding mRNAs or control mRNA were electroporated into HeLa cells. Luciferase levels were measured at 6, 24, and 48 hours after transfection (see following Table 11 and FIG. 24).

TABLE-US-00030 TABLE 11 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hours ppLuc(GC)-ag-A64-N32 33501 38979 2641 ppLuc(GC)-ag-A64-SL 28176 20364 874 ppLuc(GC)-ag-A64-U30 41632 54676 3408 ppLuc(GC)-ag-A64-G30 46763 49210 3382 ppLuc(GC)-ag-A64-PolioCL 46428 26090 1655 ppLuc(GC)-ag-A64-aCPSL 34176 53090 3338 ppLuc(GC)-ag-A64-ag 18534 18194 989 ppLuc(GC)-ag-A64-histoneSL 282677 437543 69292 ppLuc(GC)-ag-histoneSL 27597 3171 0 ppLuc(GC)-ag-A64 14339 48414 9357

[0396] Both a poly(A) sequence or the histoneSL gave rise to comparable luciferase levels. Again, the combination of poly(A) and histoneSL strongly increased the luciferase level, manifold above the level observed with either of the individual elements, thus acting synergistically. In contrast, combining poly(A) with any of the alternative sequences was without effect on the luciferase level compared to mRNA containing only a poly(A) sequence. Thus, the combination of poly(A) and histoneSL increases protein expression from mRNA in a synergistic manner, and this effect is specific.

[0397] 14.5 the Combination of Poly(A) and histoneSL Increases Protein Expression from mRNA in a Synergistic Manner In Vivo.

[0398] To investigate the effect of the combination of poly(A) and histoneSL on protein expression from mRNA in vivo, Luciferase-encoding mRNAs with different sequences 3 of the alpha-globin 3-UTR or control mRNA were injected intradermally into mice: mRNAs contained either an A64 poly(A) sequence or a histoneSL instead, or both A64 poly(A) and histoneSL 3 of the 3-UTR. Luciferase levels were measured at 16 hours after injection (see following Table 12 and FIG. 25).

TABLE-US-00031 TABLE 12 RLU at mRNA 16 hours ppLuc(GC)-ag-A64-histoneSL 38081 ppLuc(GC)-ag-histoneSL 137 ppLuc(GC)-ag-A64 4607

[0399] Luciferase was expressed from mRNA having either a histoneSL or a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL further strongly increased the luciferase level, manifold above the level observed with either of the individual elements. The magnitude of the rise in luciferase level due to combining poly(A) and histoneSL in the same mRNA demonstrates that they are acting synergistically.

[0400] The synergy between poly(A) and histoneSL was quantified by dividing the signal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals from histoneSL mRNA (?/+) plus poly(A) mRNA (+/?) (see following Table 13).

TABLE-US-00032 TABLE 13 RLU at A64 histoneSL 16 hours + + 38081 ? + 137 + ? 4607 Synergy 8.0

[0401] The factor thus calculated specifies how much higher the luciferase level from mRNA combining poly(A) and histoneSL is than would be expected if the effects of poly(A) and histoneSL were purely additive. The luciferase level from mRNA combining poly(A) and histoneSL was 8 times higher than if their effects were purely additive. This result confirms that the combination of poly(A) and histoneSL effects a markedly synergistic increase in protein expression in vivo.

[0402] 14.6 the Combination of Poly(A) and histoneSL Increases NY-ESO-1 Protein Expression from mRNA.

[0403] To investigate the effect of the combination of poly(A) and histoneSL on protein expression from mRNA, NY-ESO-1-encoding mRNAs with different sequences 3 of the alpha-globin 3-UTR were synthesized: mRNAs contained either an A64 poly(A) sequence or both A64 poly(A) and histoneSL 3 of the 3-UTR. NY-ESO-1-encoding mRNAs were electroporated into HeLa cells. NY-ESO-1 levels were measured at 24 hours after transfection by flow cytometry (see following Table 14 and FIG. 26).

TABLE-US-00033 TABLE 14 MFI at 24 hours mRNA anti-NY-ESO-1 isotype control NY-ESO-1(GC)-ag-A64-histoneSL 15600 1831 NY-ESO-1(GC)-ag-A64 1294 849

[0404] NY-ESO-1 was expressed from mRNA having only a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL strongly increased the NY-ESO-1 level, manifold above the level observed with only a poly(A) sequence.

[0405] 14.7 the Combination of Poly(A) and histoneSL Increases the Level of Antibodies Elicited by Vaccination with mRNA.

[0406] To investigate the effect of the combination of poly(A) and histoneSL on the induction of antibodies elicited by vaccination with mRNA, C57BL/6 mice were vaccinated intradermally with protamine-complexed, NY-ESO-1-encoding mRNAs with different sequences 3 of the alpha-globin 3-UTR. mRNAs contained either an A64 poly(A) sequence or both A64 poly(A) and histoneSL 3 of the 3-UTR. The level of NY-ESO-1-specific antibodies in vaccinated and control mice was analyzed by ELISA with serial dilutions of sera (see following Table 15 and FIG. 27).

TABLE-US-00034 TABLE 15 mRNA mean IgG2a[b] endpoint titer NY-ESO-1(GC)-ag-A64-histoneSL 763 NY-ESO-1(GC)-ag-A64 20

[0407] Anti NY-ESO-1 IgG2a[b] was induced by mRNA having only a poly(A) sequence. Strikingly however, the combination of poly(A) and histoneSL strongly increased the anti NY-ESO-1 IgG2a[b] level, manifold above the level observed with only a poly(A) sequence.