Artificial nucleic acid molecules for improved protein expression

Abstract

The invention relates to an artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal. The invention further relates to a vector comprising the artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal, to a cell comprising the artificial nucleic acid molecule or the vector, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.

Claims

1. A method for treating or preventing an infectious disease, the method comprising administering an RNA molecule comprising: a) at least one open reading frame (ORF) encoding an antigen from a pathogen associated with the infectious disease; and b) a 3′-untranslated region (3′-UTR) comprising at least two poly(A) sequences, wherein at least one of the poly(A) sequences comprises at least 70 adenine nucleotides, wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides, wherein the RNA molecule is administered intramuscularly.

2. The method of claim 1, wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising a poly(C) element and/or a histone stem-loop element.

3. The method of claim 1, wherein the pathogen is selected from the group consisting of a bacterial, a viral, a fungal, and a protozoan pathogen.

4. The method of claim 1, wherein at least one of the poly(A) sequences comprises at least 150 adenine nucleotides.

5. The method of claim 1, wherein at least one of the poly(A) sequences is located at the 3′ terminus of the RNA molecule.

6. The method of claim 1, wherein the 3′-UTR further comprises at least one 3′-UTR element that is not a poly(A) sequence.

7. The method of claim 1, wherein the RNA molecule further comprises a 5′-cap structure, a poly(C) sequence, a histone stem-loop, and/or an IRES motif.

8. The method of claim 7, wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 11.

9. The method of claim 1, wherein the RNA molecule further comprises a 5′-UTR and/or a promoter containing-sequence.

10. The method of claim 9, wherein the 5′-UTR comprises at least a portion of a 5′-UTR of a 5′ Terminal Oligopyrimidine Tract (TOP) gene.

11. The method of claim 1, wherein the open reading frame is at least partially G/C modified, wherein the encoded antigen sequence is not altered.

12. The method of claim 11, wherein the G/C content of the open reading frame is increased compared to a wild type open reading frame, wherein the encoded antigen sequence is not altered.

13. The method of claim 1, wherein the open reading frame is at least partially codon-optimized.

14. The method of claim 13, wherein the RNA molecule comprises at least one nucleotide analogue.

15. The method of claim 14, wherein the at least one nucleotide analogue is a modified form of uridine.

16. The method of claim 15, wherein the modified form of uridine is chemically altered by methylation.

17. The method of claim 16, wherein the modified form of uridine is a naturally occurring variant of uridine.

18. The method of claim 17, wherein the RNA molecule is associated or complexed with a cationic carrier or a polycationic carrier.

19. The method of claim 1, wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising a poly(C) element and a histone stem-loop element.

20. The method of claim 1, wherein the RNA molecule is associated with or complexed with a cationic or polycationic compound or polymeric carrier.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

(2) FIG. 1: DNA sequence (SEQ ID NO: 13) encoding the mRNA sequence, which has been used in the experiments and which comprises the sequences encoding the following elements:

(3) rpl32-PpLuc(GC)-albumin7-A64-C30-histone stem-loop.

(4) Within the DNA sequence, the sequence elements corresponding to the following elements in the mRNA are highlighted: PpLuc(GC) (ORF) in italics, rpl32 (5′-UTR) underlined and albumin7 (3′-UTR) underlined.

(5) FIG. 2: Polyadenylation of the mRNA sequence corresponding to SEQ ID NO: 13:

(6) A. Ca. 160 adenylates were added to one lot of mRNA (Lot 1). Ca. 380 adenylates were added to a different lot of mRNA (Lot 2). mRNA corresponding to SEQ ID NO: 13 was loaded onto the left lane, the respective adenylated mRNA was loaded onto the right line. A molecular size marker was loaded on the outermost lanes for size comparison (the numbers in FIG. 2A indicate the number of nucleotides comprised in the marker molecules; the same marker was used in FIG. 2B and FIG. 2C). B. Ca. 430 adenylates were added to the mRNA corresponding to SEQ ID NO: 13. C. Ca. 1000 adenylates were added to the mRNA corresponding to SEQ ID NO: 13.

(7) FIG. 3: Protein expression from polyadenylated mRNA in cultured cells:

(8) mRNA corresponding to SEQ ID NO: 13, to which ca. 160 (mRNA lot 1) or ca. 380 (mRNA lot 2) adenylates have been added by polyadenylation, was transfected into human dermal fibroblasts (HDF) and luciferase levels were measured at the indicated time points. FIG. 3 shows the results as mean RLU (relative light units)+/−SEM (standard error) for triplicate transfections.

(9) FIG. 4: Protein expression from polyadenylated mRNA after intramuscular injection into mice:

(10) 2 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 380 adenylates have been added by polyadenylation, were intramuscularly injected into mice. FIG. 4 shows the results as the median of up to 10 replicates.

(11) FIG. 5: Protein expression from polyadenylated mRNA after intramuscular injection into mice:

(12) 10 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 430 adenylates have been added by polyadenylation, were intramuscularly injected into mice. FIG. 5 shows the results as the median of up to 10 replicates.

(13) FIG. 6: Protein expression from polyadenylated mRNA after intramuscular injection into mice:

(14) 1 μg of mRNA corresponding to SEQ ID NO: 13, to which ca. 1000 adenylates have been added by polyadenylation, was intramuscularly injected into mice. FIG. 6 shows the results as the median of up to 10 replicates.

(15) FIG. 7: Induction of HA neutralizing antibodies by polyadenylated mRNA after intramuscular injection into mice:

(16) FIG. 8: DNA sequence (SEQ ID NO: 14) encoding the mRNA sequence, which has been used in the experiments and which comprises the sequences encoding the following elements:

(17) 32L4-H1N1(Netherlands2009)-HA(GC)-albumin7-A64-N5-C30-histoneSL

(18) FIG. 9: Induction of virus neutralizing titers by polyadenylated mRNA after intramuscular injection into mice.

(19) FIG. 10: DNA sequence (SEQ ID NO: 15) encoding the mRNA sequence, which has been used in the experiments and which comprises the sequences encoding the following elements:

(20) 32L4-RAVG(GC)-albumin7-A64-N5-C30-histoneSL

EXAMPLES

(21) The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

(22) 1. Preparation of DNA-Templates

(23) A vector for in vitro transcription was constructed containing a T7 promoter and a GC-enriched sequence encoding Photinus pyralis luciferase (PpLuc(GC)). The 5′ untranslated region (5′-UTR) of ribosomal protein Large 32 was inserted 5′ of PpLuc(GC). A 3′-UTR derived from human albumin (albumin7) was inserted 3′ of PpLuc(GC). Furthermore, an A64 poly(A) sequence, followed by C30 and a histone stem-loop sequence, was inserted 3′ of albumin7. The histone stem-loop sequence was followed by a restriction site used for linearization of the vector prior to in vitro transcription. mRNA obtained from this vector accordingly by in vitro transcription is designated as “rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL”.

(24) In summary, a vector was generated that comprises the sequence, which encodes the mRNA, which was used in further experiments. The DNA sequence (SEQ ID NO: 13) encoding said mRNA is shown in FIG. 1. The mRNA corresponding to said DNA sequence is characterized by the following elements:

(25) rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL

(26) Therein, the following abbreviations are used: PpLuc (GC): GC-enriched mRNA sequence encoding Photinus pyralis luciferase rpl32: 5′-UTR of human ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidine tract albumin7: 3′-UTR of human albumin with three single point mutations introduced to remove a T7 termination signal as well as a HindIII and a XbaI restriction site A64: poly(A)-sequence with 64 adenylates C30: poly(C)-sequence with 30 cytidylates histoneSL: histone stem-loop sequence according to SEQ ID NO: 11.

(27) Further constructs used in the experiments:

(28) 32L4-H1N1(Netherlands2009)-HA(GC)-albumin7-A64-N5-C30-histoneSL (SEQ ID NO: 14)

(29) 32L4-RAV-G(GC)-albumin7-A64-N5-C30-histoneSL (SEQ ID NO: 15)

(30) The templates were prepared as described for rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL.

(31) 2. In Vitro Transcription

(32) The DNA template prepared in Example 1 was linearized and transcribed in vitro using T7 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.

(33) 3. Enzymatic Adenylation

(34) RNA was reacted with E. coli poly(A) polymerase (Cellscript) using 1 mM ATP at 37° C. for 30 or 60 min. Immediately afterwards, RNA was purified using a spin column (RNeasy mini column, Quiagen). RNA was run on a gel to assess RNA extension.

(35) For vaccination experiments the mRNA was optionally complexed with protamine. mRNA complexation consisted of a mixture of 50% naked mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, naked mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution. The obtained formulated mRNA vaccine was used for in vivo experiments.

(36) 4. Protein Expression by mRNA Lipofection

(37) Human dermal fibroblasts (HDF) were seeded in 96-well plates three days before transfection at a density of 10.sup.4 cells per well. Immediately before lipofection, cells were washed in Opti-MEM. Cells were lipofected with 25 ng of PpLuc-encoding mRNA per well complexed with Lipofectamine2000. mRNA encoding Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (2.5 ng of RrLuc mRNA per well). 90 minutes after initiation of the transfection, Opti-MEM was exchanged for medium. 6, 24, 48, and 72 hours after transfection, medium was aspirated and cells were lysed in 100 μl of lysis buffer (Passive Lysis Buffer, Promega). Lysates were stored at −80° C. until luciferase activity was measured.

(38) 5. Luminescence Measurement in Cell Lysate

(39) Luciferase activity was measured as relative light units (RLU) in a Hidex Chameleon plate reader. PpLuc activity was measured at 2 seconds measuring time using 20 μl of lysate and 50 μl of luciferin buffer (Beetle-Juice, PJK GmbH). RrLuc activity was measured at 2 seconds measuring time using 20 μl of lysate and 50 μl of coelenterazin buffer (Renilla-Juice, PJK GmbH).

(40) 6. Protein Expression by Intramuscular mRNA Injection

(41) Mice were anaesthetized by intraperitoneal injection of a Ketavet and Rompun mixture. After shaving the lower leg of the animal, 2 μg of PpLuc-encoding mRNA in 20 μl of Ringer's lactate (80%) were injected intramuscularly (M. tibialis or M. gastrocnemius).

(42) 7. In Vivo Luminescence Imaging

(43) Mice were anaesthetized by intraperitoneal injection of a Ketavet and Rompun mixture. 150 μl of Luciferin solution (20 g/l) were injected intraperitoneally. 10 minutes after Luciferin injection, luminescence was recorded on an IVIS Lumina II Imaging System.

(44) Results

(45) 8.1 Additional Polyadenylation of the Artificial mRNA Increases Protein Expression from the Artificial mRNA In Vitro

(46) To investigate the effect of additional polyadenylation of the artificial mRNA on protein expression from the mRNA, the artificial mRNA was synthesized by in vitro transcription (rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL). Part of one lot of mRNA was enzymatically adenylated to add a poly(A) tail of ca. 160 adenylates (Lot 1). Part of a different lot of mRNA was enzymatically adenylated to add a poly(A) tail of ca. 380 adenylates (Lot 2) (see FIG. 2).

(47) Luciferase-encoding mRNAs were transfected into human dermal fibroblasts (HDF) in triplicate. Luciferase levels were measured at 6, 24, 48, and 72 hours after transfection. From these data, total protein expressed from 0 to 72 hours was calculated as the area under the curve (AUC) (see following Table 1 and FIG. 3).

(48) TABLE-US-00008 TABLE 1 Luciferase activity measured in human dermal fibroblasts (HDF) RLU at RLU at RLU at RLU at Poly(A) tail 6 hours 24 hours 48 hours 72 hours AUC Lot 1 incl. A64 37252 59085 29825 14612 2579000 Lot 1 incl. A64 81043 246102 89506 41308 8784000 plus ca. A160 (A224) Lot 2 incl. A64 47959 61053 23001 14053 2578000 Lot 2 incl. A64 69780 188560 69269 44478 6993000 plus ca. A380 (A444)

(49) Total Luciferase expression was identical from both mRNA lots containing an in vitro transcribed A64 sequence. The addition of ca. 160 adenylates to the 3′ end of the mRNA (resulting in a 3′-UTR comprising ca. 224 adenylates that are comprised in a poly(A) sequence) increased luciferase expression by factor 3.4. Addition of ca. 380 adenylates to the 3′ end of the mRNA (resulting in a 3′-UTR comprising ca. 444 adenyates that are comprised in a poly(A) sequence) increased luciferase expression only to a similar extent, by factor 2.7.

(50) Thus, addition of (further) adenylates to the 3′ end of the mRNA markedly increases the in vitro expression of the protein encoded by the mRNA. In particular, a 3′-UTR comprising more than 64 adenylates that are comprised in a poly(A) sequence markedly increases protein expression in vitro.

(51) 8.2 Additional Polyadenylation of the Artificial mRNA Strongly Increases Protein Expression from the Artificial mRNA after Intramuscular Injection

(52) To investigate the effect of additional polyadenylation of the artificial mRNA on protein expression from the intramuscularly injected mRNA, the artificial mRNA was synthesized by in vitro transcription (rpl32-PpLuc(GC)-albumin7-A64-C30-histoneSL). Part of this mRNA was enzymatically adenylated to add a poly(A) tail of ca. 380 adenylates.

(53) 2 μg of luciferase-encoding mRNAs were injected intramuscularly (M. tibialis or M. gastrocnemius) in BALB/c mice (10 replicates per group). In vivo luminescence was recorded the following days (see FIG. 4). From these data, total protein expressed from 0 to 15 days was calculated as the area under the curve. Luciferase was clearly expressed from intramuscularly injected mRNA containing an A64 sequence. Strikingly, however, additional polyadenylation of the artificial mRNA with 380 adenylates increased luciferase expression very strongly, raising total luciferase expressed eightfold. The magnitude of the rise in expression due to the (additional) poly(A) tail was unanticipated considering the much smaller effect observed in cultured cells. Thus, the addition of (further) adenylates comprised in a poly(A) sequence increases protein expression from intramuscularly injected mRNA very strongly to an unanticipated extent. In parallel, the effect of the additional polyadenylation of the artificial mRNA with about 430 adenylates (see FIG. 5) and about 1000 adenylates (see FIG. 6), respectively, was tested. While the both of the mRNAs that were polyadenylated with about 430 adenylates and about 1000 adenylates, respectively, led to increased protein expression as compared to non-polyadenlyated mRNA, no further increase was observed with respect to the artificial mRNA polyadenylated with 380 adenylates.

(54) 9. Vaccination with mRNA Encoding HA:

(55) Balb/c mice were vaccinated 2 times (d0 and d21) into both M. tibialis. 8 mice were vaccinated with 40 μg R2564 (naked HA mRNA), 8 animals were vaccinated with 40 μg polyadenylated R2564 (SEQ ID NO: 14; naked, polyadenylated HA), 8 animals were vaccinated with 40 μg R2630 (formulated HA mRNA) and 8 animals were vaccinated with 40 μg first polyadenylated and then formulated R2564. 8 mice injected with RiLa served as controls. Blood was collected on d35.

(56) 9.1. Hemagglutination Inhibition Assay (HI)

(57) In a 96-well plate, the obtained sera were mixed with HA H1N1 antigen (A/California/07/2009 (H1N1); NIBSC) and red blood cells (4% erythrocytes; Lohmann Tierzucht). In the presence of HA neutralizing antibodies, an inhibition of hemagglutination of erythrocytes can be observed. The lowest level of titered serum that resulted in a visible inhibition of hemagglutination was the assay result.

(58) Results:

(59) The results show that higher HI titers could be reached by polyadenylation of the mRNA. All mice treated with polyadenylated mRNA reached a level over potentially protective virus neutralizing titers (>40).

(60) 10. Vaccination with mRNA Encoding RAV G:

(61) Balb/c mice were vaccinated 2 times (d0 and d21) with 20 μg RAV-G mRNA into both M. tibialis. 8 animals were vaccinated i.m. with R2506 (SEQ ID NO: 15; naked RAV-G mRNA), 8 animals were vaccinated i.m. with polyadenylated R2506 (naked RNA) and 8 mice were injected with RiLa as controls. Blood was collected 28 days after prime. Serum was analyzed for VNTs.

(62) 10.1. Virus Neutralization Test

(63) Detection of the virus neutralizing antibody response (specific B-cell immune response) was carried out by a virus neutralisation assay. The result of that assay is referred to as virus neutralization titer (VNT). According to WHO standards, an antibody titer is considered protective if the respective VNT is at least 0.5 IU/ml. Therefore, blood samples were taken from vaccinated mice on day 28 and sera were prepared. These sera were used in fluorescent antibody virus neutralisation (FAVN) test using the cell culture adapted challenge virus strain (CVS) of rabies virus as recommended by the OIE (World Organisation for Animal Health) and first described in Cliquet F., Aubert M. & Sagne L. (1998); J. Immunol. Methods, 212, 79-87. In brief, heat inactivated sera were tested as quadruplicates in serial two-fold dilutions with respect to their potential to neutralise 100 TCID50 (tissue culture infectious doses 50%) of CVS in 50 μl of volume. Therefore, sera dilutions were incubated with virus for 1 hour at 37° C. (in humid incubator with 5% CO2) and subsequently trypsinized BHK-21 cells were added (4×105 cells/ml; 50 μl per well). Infected cell cultures were incubated for 48 hours in humid incubator at 37° C. and 5% CO2. Infection of cells was analysed after fixation of cells using 80% acetone at room temperature using FITC anti-rabies conjugate. Plates were washed twice using PBS and excess of PBS was removed. Cell cultures were scored as positive or as negative with regard to the presence of rabies virus. Negatively scored cells in sera treated wells represent neutralization of rabies virus. Each FAVN tests included WHO or OIE standard serum (positive reference serum) that served as reference for standardisation of the assay. Neutralization activity of test sera was calculated with reference to the standard serum provided by the WHO and displayed as International Units/ml (IU/m1).

(64) Results

(65) As can be seen in FIG. 9, the polyadenylated RAV-G mRNA induces higher neutralizing antibody titers, well above the WHO standard of 0.5 IU/ml.