Artificial nucleic acid molecules

Abstract

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

Claims

1. An artificial nucleic acid molecule comprising a) at least one open reading frame (ORF) encoding a pathogen antigen, tumor antigen or a human therapeutic protein, b) at least one Sac Domain-Containing Inositol Phosphatase 3 (FIG4) 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence from a human FIG4 gene 3′-UTR, and c) a poly(A) sequence having a length of 40 to 200 adenine nucleotides, wherein said ORF is heterologous to said 3′-UTR element and wherein said 3′-UTR element is positioned between said ORF and said poly(A) sequence.

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

3. The cell according to claim 2, wherein the cell is a mammalian cell.

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

5. The pharmaceutical composition according to claim 4, further comprising one or more pharmaceutically acceptable vehicles, diluents, excipients, or adjuvants.

6. A kit comprising the artificial nucleic acid molecule according to claim 1.

7. The kit according to claim 6, further comprising instructions for use, cells, an adjuvant, means for administration of a pharmaceutical composition, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable solution for dissolution or dilution of the artificial nucleic acid molecule, a vector, cells, or a pharmaceutical composition.

8. The artificial nucleic acid molecule according to claim 1, further comprising one or more of a 5′-cap structure, a 5′-UTR, a poly(C) sequence, a histone stem-loop, and an internal ribosome entry site.

9. The artificial nucleic acid molecule according to claim 8, wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 5.

10. The artificial nucleic acid molecule according to claim 8, wherein the 5′-UTR is a 5′ terminal oligopyrimidine tract UTR.

11. The artificial nucleic acid molecule according to claim 1, wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame.

12. The artificial nucleic acid molecule according to claim 1, wherein the open reading frame comprises a codon-optimized region.

13. The artificial nucleic acid molecule according to claim 1, wherein the artificial nucleic acid is an mRNA molecule.

14. The artificial nucleic acid molecule according to claim 1, wherein the open reading frame encodes a human therapeutic protein.

15. The artificial nucleic acid molecule according to claim 1, wherein the open reading frame encodes a pathogen antigen.

16. The artificial nucleic acid molecule according to claim 1, wherein the open reading frame encodes a tumor antigen.

17. The artificial nucleic acid molecule according to claim 1, comprising a sequence according to SEQ ID NO:1 or 2.

18. A vector comprising a) an open reading frame encoding a pathogen antigen, tumor antigen or a human therapeutic protein, b) at least one Sac Domain-Containing Inositol Phosphatase 3 (FIG4) 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence from a human FIG4 gene 3′-UTR, and c) a poly(A) sequence having a length of 40 to 200 adenine nucleotides, wherein said ORF is heterologous to said 3′-UTR element and wherein said 3′-UTR element is positioned between said ORF and said poly(A) sequence.

19. A purified artificial mRNA molecule comprising, from 5′ to 3′, a) at least one open reading frame (ORF) encoding a pathogen antigen, tumor antigen or a human therapeutic protein, b) at least one Sac Domain-Containing Inositol Phosphatase 3 (FIG4) 3′-untranslated region element (3′-UTR element) comprising a nucleic acid sequence from a human FIG4 gene 3′-UTR, and c) a poly(A) sequence having a length of 40 to 200 adenine nucleotides, wherein said ORF is heterologous to said 3′-UTR element.

20. The purified artificial mRNA molecule according to claim 19, wherein the open reading frame encodes a human therapeutic protein.

21. The purified artificial mRNA molecule according to claim 19, wherein the open reading frame encodes a pathogen antigen.

22. The purified artificial mRNA molecule according to claim 19, wherein the open reading frame encodes a tumor antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following Figures, Sequences and Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(2) FIG. 1: Effect of the human FIG4 3′-UTR on luciferase expression from an artificial mRNA. Therein, the mRNA comprising the human FIG4 3′-UTR is an mRNA according to the present invention. It comprises—in 5′-to-3′-direction—a 32L4 5′-UTR5′-UTR, a GC-enriched sequence encoding Photinus pyralis luciferase, a 3′-UTR element according to SEQ ID No. 2, a poly(A) sequence having a length of 64 adenines and a poly(C) sequence of 30 cytidine nucleotides. A markedly extended protein expression from the artificial mRNA containing the human FIG4 3′-UTR corresponding to SEQ ID No. 2 is observable compared to the mRNA lacking the FIG4 3′ UTR. Data are graphed as RLU±SD (mean of relative light units±standard deviation) for triplicate transfections. RLU are summarized in Example 5.1.

(3) The following abbreviations are used: PpLuc (GC): GC-enriched mRNA sequence coding for Photinus pyralis luciferase A64: poly(A)-sequence with 64 adenylates C30: poly(C)-sequence with 30 cytidylates hSL: a histone stem-loop sequence taken from (Cakmakci, Lerner, Wagner, Zheng, & William F Marzluff, 2008. Mol. Cell. Biol. 28(3):1182-94). 32L4: 5′-UTR of human ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidine tract fig4: 3′-UTR of human fig4 (Homo sapiens FIG4 homolog, SAC1 lipid phosphatase domain containing (S. cerevisiae) (FIG4), mRNA).

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

(5) FIG. 3: mRNA sequence of 32L4-PpLuc(GC)-fig4-A64-C30-hSL (SEQ ID NO: 7). The 3′-UTR is derived from human Fig4 transcript. The PpLuc(GC) ORF is highlighted in italics, the 3′ UTR is underlined.

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

(7) FIG. 5: mRNA sequence of PpLuc(GC)-fig4-A64-C30-hSL (SEQ ID NO: 10). The 3′-UTR is derived from human Fig4 transcript. The PpLuc(GC) ORF is highlighted in italics, the 3′ UTR is underlined.

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

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

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

EXAMPLES

(11) 1. Preparation of DNA-Templates

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

(13) This vector was modified to include the 3′-UTR of the human fig4 transcript 3′ of the PpLuc ORF.

(14) mRNAs obtained from these vectors by in vitro transcription are designated as:

(15) 32L4-PpLuc(GC)-A64-C30-hSL (FIG. 2; SEQ ID NO: 8)

(16) 32L4-PpLuc(GC)-fig4-A64-C30-hSL (FIG. 3; SEQ ID NO: 7)

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

(18) Also this vector was modified to include the 3′UTR of the human fig4 transcript 3′ of the PpLuc ORF.

(19) mRNAs obtained from these vectors by in vitro transcription are designated as:

(20) PpLuc(GC)-A64-C30-hSL (FIG. 4; SEQ ID NO: 9)

(21) PpLuc(GC)-fig4-A64-C30-hSL (FIG. 5; SEQ ID NO: 10).

(22) 2. In Vitro Transcription

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

(24) 3. Luciferase Expression by mRNA Lipofection

(25) Human HeLa cells were seeded in 96 well plates at a density of 1×10.sup.4 cells per well medium (RPMI 1640 medium with L-glutamine and 25 mM Hepes (Lonza, Basel, Switzerland) to which 10% FCS, 1% Pen/Strep, 1% Glutamine were added). The following day, cells were washed in Opti-MEM® I Reduced Serum Medium (Gibco, Life Technologies, Carlsbad, Calif., USA) and then transfected with 12.5 ng per well of Lipofectamine2000-complexed PpLuc-encoding mRNA in Opti-MEM. Untransfected cells served as control. mRNA coding for Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (1 ng of RrLuc mRNA per well). 90 minutes after start of transfection, Opti-MEM was exchanged for medium. 24, 48, 72 hours after transfection, medium was aspirated and cells were lysed in 100 μl of Passive Lysis buffer (Promega). Lysates were stored at −80° C. until luciferase activity was measured.

(26) 4. Luciferase Measurement

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

(28) 5. Luciferase Expression by Intradermal mRNA Injection

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

(30) Results

(31) 6.1 Protein Expression from mRNA Containing a FIG4 3′-UTR is Prolonged

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

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

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

(35) Luciferase was clearly expressed from mRNA lacking the FIG4 3′-UTR. The FIG4 3′-UTR significantly extended luciferase expression.

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

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

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

(39) 6.2 Protein Expression from mRNA Containing a FIG4 3′UTR is Increased after Injection in Mice.

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

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

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

(43) In comparison with the albumin7 3′-UTR, the fig4 3′-UTR significantly increased the expression of luciferase protein from the respective mRNA.