Ribonucleic acid construct capable of inducing an immune response, as well as pharmaceutical composition and kit comprising same

Abstract

The present invention relates to a ribonucleic acid construct capable of inducing an immune response, comprising a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5´-end of at least one strand of said sequence, and a single-stranded overhang at at least one strand of said sequence, wherein the single-stranded overhang comprises at least one guanosine. The present invention also relates to the use of said construct as a vaccine or as an immune adjuvant as well as to the ribonucleic acid construct for use as a medicament or for use in a method of treatment or prevention of a disease, specifically for use in a method of treatment or prevention of viral infections or cancer. The present invention further relates to a pharmaceutical composition and kit comprising said ribonucleic acid construct.

Description

RIBONUCLEIC ACID CONSTRUCT CAPABLE OF INDUCING AN IMMUNE RESPONSE, AS WELL AS PHARMACEUTICAL COMPOSITION AND KIT COMPRISING SAMECROSS-REFERENCE TO RELATED APPLICATIONS AND CLAIM TO PRIORITY

[0001] This application claims priority to European patent application with Application No. 23205 742.2, filed 25 October 2023 at the European Patent Office, wherein the disclosure thereof is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to a ribonucleic acid (RNA) construct capable of inducing an immune response, comprising a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of at least one strand of said sequence, and a single-stranded overhang at at least one strand of said sequence, wherein the single-stranded overhang comprises at least one guanosine. Said construct may be able to bind to and activate retinoic acid-inducible gene I (RIG-I). The present invention also relates to said ribonucleic acid construct for use as a medicament, for use in a method of treatment or prevention of a disease, for use in a method of infiltrating immune cells, preferably T cells, and for use in a method of turning cold into hot tumours. The present invention also provides the use of said ribonucleic acid construct in the manufacture of a medicament for the treatment or prevention of a disease, a method of treating a disease comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct to a subject in need thereof, and a method of inducing an immune response. Additionally, the present invention also relates to a respective pharmaceutical composition and further to a respective kit for use in medicine, each comprising the ribonucleic acid construct.BACKGROUND OF THE INVENTION

[0003] Retinoic-acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) that detects the presence of viral genomic material, specifically single- or double-stranded RNAgenomes and induces a type-1 interferon response, thus activating innate immune responses in proximal cells. This allows for pre-emptive warning of viral infection that may limit its further spread to other cells or tissues. It is therefore desirable to identify artificial RIG-1 ligands that may be used to activate the innate immune system, thereby triggering therapeutic and / or prophylactic responses that can assist in the treatment of diseases, such as cancer, or in the treatment of viral infections, either of a current infection or as a prophylactic treatment to reduce the risk of future infection.

[0004] Artificial RIG-1 ligands are typically embodied by ribonucleic acid (RNA) constructs that contain the requisite molecular motifs to activate RIG-1 (for example, see EP 4015636 A1 and EP 2123757 A1). Such RNA ligands can vary in precise composition, reflecting the uncertainty or diversity of essential molecular motifs needed for RIG-I activation.

[0005] Thus, there is a high need for an improvement in efficiency of construct design, whilst retaining potency as a RIG-I agonist, and in transfection capability, as well as enabling the use of such a construct in methods for use in the treatment or prevention of a disease.SUMMARY OF THE INVENTION

[0006] The construct of the present invention results in high-levels of RIG-I activation and subsequent IFN production, whilst retaining a simple construction and comprising a relative short length compared to constructs described in the prior art. In addition to the simplicity of design, the construct of the present invention may simplify the synthesis of RIG-I ligands with the ability to penetrate cells in the absence of normally requisite transfection reagents.

[0007] The present invention provides a ribonucleic acid construct capable of inducing an immune response, comprising- a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of at least one strand of said sequence, and- a single-stranded overhang at at least one strand of said sequence, wherein the singlestranded overhang comprises at least one guanosine.

[0008] Preferably, said ribonucleic acid construct may be capable of inducing an immune response via activating retinoic-acid inducible gene I (RIG-I). More preferably, it may induce an anti-viral immune response, which may be preferably via induction of interferon expression and release.

[0009] It is preferred that the double-stranded ribonucleic acid sequence of said ribonucleic acid construct has a length of 8 to 15 ribonucleotides, more preferably a length of 8 to 12 ribonucleotides, even more preferably a length of 10 to 12 ribonucleotides, and even more preferably a length of 11 ribonucleotides.

[0010] It is further preferred for the ribonucleic acid construct of the present invention that the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which also comprises the single-stranded overhang. However, it is also contemplated for the ribonucleic acid construct of the present invention that the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which does not comprise the single-stranded overhang. However, the present invention also comprises that the double-stranded ribonucleic acid sequence may comprise a triphosphate at the 5'-end of both stands.

[0011] It is also preferred for the ribonucleic acid construct of the present invention that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of at least 3 nucleosides, more preferably a length of 3 to 15 nucleosides, more preferably a length of 3 to 10 nucleosides, more preferably a length of 3 to 8 nucleosides, more preferably a length of 3 to 5 nucleosides. Said overhang may comprise at least two guanosines, preferably at least three guanosines, more preferably three guanosines, and even more preferably three consecutive guanosines. It is also preferred that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises or consists of adenosine-adenosine-guanosine- guanosine-guanosine (AAGGG).

[0012] For the ribonucleic acid construct of the present invention, the double-stranded ribonucleic acid sequence may be a blunt-ended ribonucleic acid sequence (apart from the single-stranded overhang comprising at least one guanosine). But the present invention also comprises that the double-stranded ribonucleic acid sequence comprises a further overhang, preferably a further overhang at the strand not comprising the singlestranded overhang comprising at least one guanosine. For example, that further overhang is a single-stranded RNA-overhang or an overhang with a fluorophore or a lipophilic substitution.

[0013] It is preferred that the double-stranded ribonucleic acid sequence is connected with the single-stranded overhang via a linker moiety L. That linker moiety L may be a nucleoside-linker or a hydrocarbon-based linker. Preferably, the hydrocarbon-based linker comprises 3 to 24 main chain carbon atoms. More preferably, the hydrocarbon-based linker may comprise 6 to 18 main chain carbon atoms. In some embodiments, the linker moiety L is a nucleoside-linker, preferably a nucleoside-linker having a length of at least 2 nucleosides, more preferably a nucleoside-linker having a length of 2 to 10 nucleosides.For example, the nucleoside-linker may be an adenosine-linker, preferably with at least 2 adenosines, more preferably with 2 adenosines.

[0014] In some embodiments, the linker moiety L may be a hydrocarbon-based linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. Preferably, the linker-moiety L may be a polyalkylene-glycol-linker according to Formula IIFormula II, wherein n is an integer in the range of 3 to 8, more preferably in the range of 5 to 7. It is even more preferred that the linker moiety L is a polyalkylene-glycol-linker according to Formula II and wherein n is 6.

[0015] In some embodiments of the ribonucleic acid construct of the present invention, the ribonucleic acid construct comprises at least one modification(s). Preferably, the at least one modification(s) is / are selected from the group consisting of O-methyl-group- modification(s), preferably 2'-O-methyl-group-modification(s), O-methoxyethyl- modification(s), preferably 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s) or 2'-deoxy-2'-fluoro-group modification(s), phosphoro-thioate-modification(s), 2'-deoxy-group-modification(s), locked nucleic acid (LNA)-modification(s), and internucleoside linkage-modification(s), preferably phosphothioate-, thiophosphate-, phosphoroselenate- and selenophosphateinternucleoside linkage-modification(s). For example, the phosphorothioate- modification(s) may be phosphorothioate-backbone-modification(s) between two nucleotides.

[0016] In some embodiments, the ribonucleic acid construct is capable of penetrating permeabilized cells without the presence of a transfection reagent.

[0017] The present invention additionally provides the ribonucleic acid construct according to the present invention for use as a medicament. The present invention further provides the ribonucleic acid construct according to the present invention for use in a method of treatment or prevention of a disease.

[0018] The present invention additionally provides the ribonucleic acid construct according to the present invention for use in a method of infiltrating immune cells, preferably T cells.

[0019] The present invention further provides the ribonucleic acid construct according to the present invention for use in a method of turning cold tumours into hot tumours.

[0020] In one aspect, the present invention also provides the use of the ribonucleic acid construct according to the present invention as a vaccine, preferably wherein the vaccine further comprises an mRNA-sequence.

[0021] In another aspect, the present invention provides the use of the ribonucleic acid construct according to the present invention as an immune adjuvant.

[0022] The present invention also comprises the use of the ribonucleic acid construct according to the present invention in the manufacture of a medicament for the treatment or prevention of a disease.

[0023] In a further aspect, the present invention provides a pharmaceutical composition comprising the ribonucleic acid construct according to the present invention.

[0024] The present invention also provides the ribonucleic acid construct according to the present invention for use in a method of inducing an immune response. The ribonucleic acid construct according to the present invention may be an antigen recognizing construct or part of an antigen recognizing construct or may be comprised in an antigen recognizing construct. Preferably, the antigen recognizing construct may be an antibody, or fragment thereof, or a T cell receptor (TCR), or fragment thereof, or a chimeric antigen receptor (CAR), or a fragment thereof. For example, the immune response may be induced by cytokine production.

[0025] In a further aspect, the present invention also provides a method of treating a disease, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to the present invention to a subject in need thereof.

[0026] In another aspect, the present invention also provides a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to the present invention to a subject in need thereof.

[0027] The present invention also provides, in a further aspect, a kit for use in medicine comprising the ribonucleic acid construct according to the present invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings described in the following.

[0029] Figure 1 shows that “GI2” (SEQ ID NOs: 1 + 2), a ribonucleic acid construct according to the present invention, is a potent inducer of IFN expression in human monocytes. THP1-Dual-WT cells were transfected with either “GI2” (SEQ ID NOs: 1 +2) (dark grey, according to the present invention) or a construct named “Superoligo” (SEQ ID NOs: 49 + 50) (light grey, highly-modified 5’-triphosphate 23 bp dsRNA), in complex with Lipofectamine 2000 and were administered at 3 concentrations (0.01 , 1 and 100 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant, with comparable levels of IFN-expression induced by each construct at all 3 concentrations applied. The results represent two experiments and mean ± SEM is shown.

[0030] Figure 2 shows that the ribonucleic acid construct “GI2” (SEQ ID NOs: 1 + 2) according to the present invention comprises superior IFN-inducing effect compared to other commonly used pure ribonucleotide RIG-I ligands. IFN-expression was quantified via luciferase activity in cultured monocytes following application of “GI2” (SEQ ID NOs: 1 + 2) (dark grey), IVT4 (SEQ ID NO: 51) (light grey, 56 bp long RNA produced via in vitro transcription with a GAAA-loop and a 26 bp long dsRNA part), or GFP2 (SEQ ID NOs: 52 + 53) (white with black border, synthetic triphosphate 24 bp dsRNA). After 18 h exposure, “GI2” (SEQ ID NOs: 1 + 2) outperforms both constructs at all 3 concentrations applied (0.01 , 1 and 100 ng / well) after Lipofectamine 2000 transfection. The results represent two experiments and mean ± SEM is shown.

[0031] Figure 3 shows that “GI2” (SEQ ID NOs: 1 + 2) has a substantially stronger IFN- inducing effect compared to recently published RNA-based RIG-I ligand, RNA-1 (SEQ ID NOs: 54 + 55). RNA-1 (SEQ ID NOs: 54 + 55) (light grey) is a pure dsRNA construct comprising at least 20 bp and a sense strand: 5’-C, and antisense strand: 3’-GGG. Constructs of either “GI2” (SEQ ID NOs: 1 + 2) (dark grey) or RNA-1 (SEQ ID NOs: 54 + 55) were applied at 3 concentrations and luciferase intensity quantified, demonstrating that “GI2” (SEQ ID NOs: 1 + 2) is substantially more powerful in inducing IFN-expression at all 3 concentrations following a 18 h exposure. The results represent two experiments and mean ± SEM is shown.

[0032] Figure 4 shows that the IFN-stimulating capacity of “GI2” (SEQ ID NOs: 1+ 2) is independent of RNA nucleotide sequence. Three constructs differing in nucleotide sequence of 11 bp dsRNA, but each comprising a 5’-triphosphate, a 3’-guanosine-richoverhang with adenosine linker, were applied to THP1 Dual wt. Each construct (“GI2” (SEQ ID NOs: 1 + 2) in dark grey, “GI3” (SEQ ID NOs: 3 + 4) in light grey, and “GI4” (SEQ ID NOs: 5 + 6) in white with black border) induced comparable levels of IFN-expression, indicating that the ability of the described construct to induce IFN-expression does not depend on precise sequence of nucleotides as long as key structural elements are present - in this example, a 5’-triphosphate and 3’-guanosine-rich overhang. This demonstrates that “GI2” (SEQ ID NOs: 1 + 2) is a RIG-I ligand that - also with a rather short / minimal length - contains the essential molecular motifs required for RIG-l-mediated IFN responses. The results represent three experiments and mean ± SEM is shown.

[0033] Figure 5 shows that “GI4” (SEQ ID NOs: 5 + 6) shows superior IFN-inducing effect compared to 5’-triphosphorylated hairpin RNA constructs. Hairpin-containing, blunt-ended RNA is a widely described RIG-I ligand. Two such constructs, BM65 (SEQ ID NO: 56) (light grey) and IVT4 (SEQ ID NO: 51) (white with black border) were applied at 3 concentrations and subsequent IFN-expression was contrasted with “GI4” (SEQ ID NOs: 5 + 6) (dark grey), which does not contain a hairpin, at each concentration. “GI4” (SEQ ID NOs: 5 + 6) can be seen to induce substantially greater levels of IFN expression versus both hairpin-containing constructs at all 3 concentrations following 18 h exposure to the constructs. The results represent two experiments and mean ± SEM is shown.

[0034] Figure 6 shows that “GI2” (SEQ ID NOs: 1 + 2), when modified through three 2’- O-Me-modification(s) (SEQ ID NOs: 15 + 16) as shown in the scheme, can penetrate cells and activate RIG-I in the absence of a lipid based transfection reagent. 20 x 106THP1 Dual wt cells were incubated with 0.1 pg Phorbol-12-myristate-13-acetate (PMA, stock 1 pg / pL) in 20 mL RPMI Medium overnight at 37°C and 5 % CO2 in a 10 cm diameter dish. The cells were washed twice with PBS, transferred in a 96-well-plate with 8 x 104cells / well in 100 pL RPMI medium and incubated overnight at 37°C and 5 % CO2. The medium was removed and the cells were incubated for 30 minutes with 100 ng / well of the assigned RNA dissolved in 50 pL Digitonin containing buffer (50 mM HEPES, pH 7.0, 100 mM KCI, 3 mM MgCh, 0.1 mM DTT, 85 mM sucrose, 0.2 % BSA, 1 mM ATP, 10 pg / mL Digitonin). The RNA containing solution was removed and 100 pL RPMI medium was added. 18 h after incubation at 37°C and 5 % CO2, the induced IFN-a was significantly higher for the modified “GI2” (SEQ ID NOs: 15 + 16). The results represent two experiments and mean ± SEM is shown.

[0035] Figure 7 shows that lengthening of the guanosine-rich overhang leads to multimerization without improved IFN-induction capability. The guanosine-rich overhang, which typically comprises 3 consecutive guanosines, of the GI2 construct (SEQ ID NOs: 1 + 2) (black) was modified to either lack a guanosine-rich overhang (SEQ ID NOs: 57 + 2) (striped bar) or else to contain a guanosine-rich overhang comprising either 4 (SEQ IDNOs: 58 + 2) (dark grey) or 6 (SEQ ID NOs: 59 + 2) (light grey) sequential guanosine nucleotides. Figure 7A shows that all 4 constructs were loaded onto a 15% native gel. Constructs containing overhangs longer than 3 guanosine residues in length resulted in the observation of slow-running band in the gel, indicating a higher molecular weight and strongly indicating construct multimerization. Figure 7B shows that whilst lack of overhang resulted in a near total loss of IFN induction at all 3 concentrations applied, overhangs longer than 3 consecutive guanosine nucleotides in length did not improve, or substantially worsen, IFN induction capability at any concentration. THP1-Dual-WT cells were transfected with the four constructs in complex with Lipofectamine 2000 and were administered at 3 concentrations (0.01 , 1 and 100 ng / well). The results represent two experiments and mean ± SEM is shown.

[0036] Figure 8 shows that “GI2” (SEQ ID NOs: 1 + 2) injection induces an antiviral response in vivo. C57BL / 6 mice were intravenously injected with 12.5 pg of an RNA construct complexed with in vivo jetPEI, a transfection reagent. The following constructs were injected: CA21 (SEQ ID NO: 60) (light grey, n = 8 mice, negative control), IVT4 (SEQ ID NO: 51) (dark grey, n = 8 mice), “GI2” unmodified (SEQ ID NOs: 1 + 2) (black, n = 4 mice), and “GI2” modified (SEQ ID NOs: 44 + 42) (striped grey, n = 4 mice). Figure 8A shows 6 and 24 hours post-injection, sera were collected from mice and the concentration of IFN-a measured via ELISA. At 6 hours, both versions of “GI2” and IVT4 elicited a comparable level of IFN-a production, with CA21-elicited levels measuring substantially lower. By 24 hours, for all 4 constructs, IFN-a levels had fallen to a range comparable to IFN-a levels present in untreated sera. Figures 8B and 8C show that interferon-stimulated gene expression was determined in the spleen of mice. Expression was quantified by qPCR relative to murine tbk1 expression.

[0037] Figure 9 shows that “GI2-3M” (corresponds to SEQ ID NO: 15, the triphosphategroup was not displayed in the sequence listing, “GI2-3M” was used as sense-strand), combined with antisense strands (each of SEQ ID NOs: 16 to 26) bearing different lipophilic groups, which are ribonucleic acid constructs according to the present invention, are inducers of IFN expression in human monocytes. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown. GI2-3M + GI2-3M-AS corresponds to SEQ ID NO: 15 + SEQ ID NO: 16, GI2-3M + 5'-Chol-C6 corresponds to SEQ ID NO: 15 + SEQ ID NO: 17, GI2-3M + 5'-Chol-Pro corresponds to SEQ ID NO: 15 + SEQ ID NO: 18, GI2-3M + 5'-Teg-Chol corresponds to SEQ ID NO: 15 + SEQ ID NO: 19, GI2-3M + 5'-Palmitate corresponds to SEQ ID NO: 15 + SEQ ID NO:20, GI2-3M + 5'-Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 21 , GI2-3M + 5'- Tocopherol-C8 corresponds to SEQ ID NO: 15 + SEQ ID NO: 22, GI2-3M + 5'-lbu corresponds to SEQ ID NO: 15 + SEQ ID NO: 23, GI2-3M + 3'-Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 24, GI2-3M + 3'-lbu corresponds to SEQ ID NO: 15 + SEQ ID NO: 25 and GI2-3M + 3'-GalNac corresponds to SEQ ID NO: 15 + SEQ ID NO: 26.

[0038] Figure 10 shows that transfection of air liquid interface A549 cells with “GI2-3M” (SEQ ID NO: 15), combined with antisense strands (each of SEQ ID NOs: 27 to 31) bearing different lipophilic groups, which are ribonucleic acid constructs according to the present invention, enhance interferon-stimulated genes (RSAD2, IFI44, ISG15). Repetitive stimulation has been performed at day 0 and day 2 with 5 pg of the indicated RNA. At day 4, cells have been harvested and qPCR has been performed. Gene upregulation is shown as 2-AActrelative to the 10 mM HEPES control and the house keeping gene GAPDH. The results represent two experiments and mean ± SEM is shown. GI2-3M + Chol-C6 corresponds to SEQ ID NO: 15 + SEQ ID NO: 27, GI2-3M + Chol-Pro corresponds to SEQ ID NO: 15 + SEQ ID NO: 28, GI2-3M + Chol-Teg corresponds to SEQ ID NO: 15 + SEQ ID NO: 29, GI2-3M + Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 30 and GI2-3M + Ibuprofen corresponds to SEQ ID NO: 15 + SEQ ID NO: 31.

[0039] Figure 11 shows that different 2'-modified ribonucleic acid constructs according to the present invention (see various combinations of SEQ ID NOs: 32 to 38; namely SEQ ID NOs: 15 + 27, SEQ ID NOs: 15 + 28, SEQ ID NOs: 15 + 29, SEQ ID NOs: 15 + 30 and SEQ ID NOs: 15 + 31) are inducers of IFN expression in human monocytes. THP1-Dual- WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown. GFP2 11 AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 33, GFP2 11 AA GGG + 3F AS corresponds to SEQ ID NO: 32 + SEQ I D NO: 34, GFP2 11 3F AA GGG + GFP2 11 A corresponds to SEQ ID NO: 35 + SEQ ID NO: 33, GFP2 11 3F AA GGG + 3F AS corresponds to SEQ ID NO: 35 + SEQ ID NO: 34, GFP2 11 AA GGG + SO 3M AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 36, SO 11 3M AA GGG + SO 3M AS corresponds to SEQ ID NO: 61 + SEQ ID NO: 36, SO 11 3M AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 61 + SEQ ID NO: 36, GFP2 11 AA GGG + 3L AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 38, GFP2 11 3L AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 37 + SEQ ID NO: 33 and GFP2 11 3L AA GGG + 3L AS corresponds to SEQ ID NO: 37 + SEQ ID NO: 38.

[0040] Figure 12 shows that different modified ribonucleic acid constructs according to the present invention (various combinations of SEQ ID NOs: 39 - 46, namely SEQ ID NOs:39 + 40, 41 + 42, 41 + 43, 44 + 42, 45 + 43, 45 + 42 and 41 + 46) are inducers of IFN expression in human monocytes. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown. pppGI2-AA-GGG + GI2-AS corresponds to ppp- SEQ ID NO: 39 + SEQ ID NO: 40, pppGI2-AA-3M + GI2-AS-3M corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 42, pppGI2-AA-3M + GI2-AS-3M_2dT corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 43, pppGI2-mod1 + GI2-AS-3M corresponds to ppp-SEQ ID NO: 44 + SEQ ID NO: 42, pppGI2-3M-7T + GI2-AS-3M2T corresponds to SEQ ID NO: 45 + SEQ ID NO: 43, pppGI2-3M-7T + GI2-AS-3M corresponds to ppp-SEQ ID NO: 45 + SEQ ID NO. 42 and pppGI2-AA-3M + GI2-AS-3M2T corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 46.

[0041] Figure 13 shows that highly modified ribonucleic acid constructs according to the present invention (see SEQ ID NOs: 1 + 2 and SEQ ID NOs: 47 + 48) are inducers of IFN expression in human monocytes. pppGI2-AA-GGG + GI2-AS corresponds to SEQ ID NO: 1 + SEQ ID NO: 2 and pppSO-11-3F-dG + SO-3F-2xDNA-AS corresponds to SEQ ID NO: 47 + SEQ ID NO: 48. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0042] Figure 14 shows that ribonucleic acid constructs with different covalently linked GGG-overhangs according to the present invention (see SEQ ID NOs: 1 + 2, SEQ ID NOs: 49 + 2, and SEQ ID NOs: 50 + 2) are inducers of IFN expression in human monocytes. “GI2: AA-GGG” corresponds to SEQ ID NO: 1 + SEQ ID NO: 2, “GI2: GGG” corresponds to SEQ ID NO: 62 + SEQ ID NO: 2, “GI2: HEG GGG” corresponds to SEQ ID NO: 63 + SEQ ID NO: 2. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 3 concentrations (100, 1 and 0.01 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0043] Figure 15 shows that ribonucleic acid constructs with different G-content in the overhang of the strand not comprising the 5’-ppp-end according to the present invention (see SEQ ID NOs: 1 + 2, SEQ ID NOs: 64 + 66, and SEQ ID NOs: 64 + 67) are inducers of IFN expression in human monocytes. “GI2” corresponds to SEQ ID NO: 1 + SEQ ID NO: 2, “GI2 AS: AA GGG” corresponds to SEQ ID NO: 64 + SEQ ID NO: 65, “GI2 AS: AAGG” corresponds to SEQ ID NO: 64 + SEQ ID NO: 66, “GI2 AS: AA G” corresponds to SEQ ID NO: 64 + SEQ ID NO: 67. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 3 concentrations (100, 1 and 0.01 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.DETAILED DESCRIPTION OF THE INVENTION

[0044] As indicated above, the following invention describes a RIG-I ligand, which may have a small length and may be capable of penetrating permeabilized cells without the presence of a transfection reagent, e.g. a lipophilic transfection reagent, whilst it provides RIG-I stimulation.

[0045] Ribonucleic acid construct

[0046] Thus, in a first aspect, the present invention provides a ribonucleic acid construct capable of inducing an immune response, comprising- a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of at least one strand of said sequence, and- a single-stranded overhang at at least one strand of said sequence, wherein the singlestranded overhang comprises at least one guanosine.

[0047] As used herein, "inducing an immune response" may mean initiating or causing an increase in one or more of B-cell activation, T-cell activation, natural killer cell activation, activation of antigen presenting cells (e.g. B cells, dendritic cells, monocytes and macrophages), cytokine production, chemokine production, specific cell surface marker expression, in particular, expression of co-stimulatory molecules. In one aspect, such an immune response may involve the production of type I IFN, in particular IFN-a, in cells such as PDC (plasmacytoid dendritic cells) and / or monocytes.

[0048] It is preferred for said ribonucleic acid construct according to the present invention that it is capable of inducing an anti-viral immune response.

[0049] As used herein, "anti-viral response" refers to the response by a cell, tissue or organism upon infection by a virus with the purpose of eliminating or incapacitating the virus. Typical anti-viral responses include, but are not limited to, type I IFN, MIP1-a, MCP, RANTES, IL-8, IL-6, IP-10, and IFN-a production.

[0050] It is also preferred for the ribonucleic acid construct of the present invention that it is capable of inducing interferon (IFN)-production, preferably of inducing type l-IFN production.

[0051] “Interferons” (IFN) are a group of signalling proteins that act as extracellular ligands at interferon receptors, thereby activating innate immune system transcription pathways that boost antiviral defences. “Type l”-interferons are a subgroup of IFN, comprising 13 members that activate the IFN-a receptor and may be produced, although not exclusively, by plasmacytoid dendritic cells, fibroblasts, leukocytes, and endothelial cells.

[0052] The ribonucleic acid construct according to the present invention may be capable of activating retinoic acid-inducible gene I (RIG-I). Further, the ribonucleic acid construct according to the present invention may be capable of binding the receptor of retinoic acidinducible gene I (RIG-I).

[0053] “Retinoic acid-inducible gene (RIG-I)” is an innate immune surveillance protein and member of the pattern recognition receptor family, which detects the intracellular presence of viral genomic RNA, both single- and double-stranded, via common molecular motifs found in viral genomes. Activation of RIG-I is an important step in innate immune activation in response to the presence of certain viruses. RIG-I activation initiates downstream signalling pathways that ultimately trigger expression and release of, amongst other immune mediators, interferon(s). Interferon release triggers further innate immune activation in proximal cells, allowing them to prepare an antiviral response.

[0054] Due to the role of RIG-I in triggering innate immune responses, it is an attractive target for both prophylactic anti-viral treatments, vaccine adjuvants, and anti-cancer treatments. Identifying and optimizing minimal RIG-I ligands has therefore been an important focus of developments in this field in recent years, accelerated by the worldwide SARS-CoV2 pandemic, a coronavirus who’s genome is recognized by RIG-I.

[0055] Early work identified RIG-I activation by in vitro transcribed dsRNA greater or equal to 50 bp in length or by synthetic blunt-ended short double-stranded RNAs 21-27 bp in length.

[0056] Prior literature reported a number of structurally different RNA molecules as a ligand for RIG-I, often bearing different key molecular motifs. The lack of clarity with regard to those motifs means there is and was no consensus as to the critical molecular motif for recognition by, and subsequent activation of, RIG-I.

[0057] The inventors of the present invention identified the ribonucleic acid construct as described herein, which provides RIG-I activation, as demonstrated e.g. through application to primary monocyte cultures using interferon (IFN)-a production as a readout of RIG-I activation efficacy.

[0058] “RIG-1 activation”, as used herein, refers to the binding of RIG-1 to a suitable ligand, typically viral genomic RNA, the subsequent displacement of the inhibitory caspase activation and recruitment domains (CARDs) of RIG-1, the oligomerization of multiple RNA- bound RIG-1 monomers, and the interaction between oligomerized RIG-1 CARD domains and mitochondrial antiviral-signalling protein (MAVS), which induces gene expression, most importantly expression of type I IFNs, via kinase-based signalling pathways.

[0059] It is preferred for the ribonucleic acid construct of the present invention that the double-stranded ribonucleic acid sequence has a length of 8 to 15 ribonucleotides. It is more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 8 to 12 ribonucleotides. It is even more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 10 to 12 ribonucleotides. It is even more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 11 ribonucleotides.

[0060] Using, for example, TH P1 -dual cells, which allow for indirect monitoring of IFN production via an interferon response factor (IRF)-linked luciferase reporter gene expression system, it was demonstrated that the ribonucleic acid construct of the present invention can stimulate the IFN expression, despite it may comprise only 11 nucleotides in length (see Figure 1 , example 1 , Figure 2 and example 2). This finding was surprising, as prior art suggests that nucleotides of at least 20 bp in length are needed for RIG-I activation.

[0061] The inventors of the present invention demonstrate that, despite it’s short length, RIG-I activation efficacy does not depend on precise RNA nucleotide sequence, outside of the presence of a guanosine-rich single-stranded overhang, for example.

[0062] Triphosphate

[0063] A triphosphate group, also named as “ppp” here within, refers to three phosphate groups (PO ') sequentially bound to the ribose sugar of a nucleotide molecule.

[0064] In some embodiments of the ribonucleic acid construct of the present invention, it is preferred that the at least one triphosphate is at the 5'-end of the strand of the doublestranded ribonucleic acid sequence which also comprises the single-stranded overhang.

[0065] However, it is also comprised in the present invention that the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which does not comprise the single-stranded overhang.

[0066] It is also preferred for the ribonucleic acid construct of the present invention that the double-stranded ribonucleic acid sequence comprises a triphosphate at the 5'-end of both strands. That means there is a triphosphate at the 5'-end of each strand of said double-stranded ribonucleic acid sequence of the construct according to the present invention.

[0067] Single-stranded overhang

[0068] The ribonucleic acid construct of the present invention comprises a single-stranded overhang at at least one strand of said double-stranded ribonucleic acid sequence, wherein the single-stranded overhang comprises at least one guanosine.

[0069] The term “overhang” is used interchangeably herein with the term “single-stranded overhang” and refers to a non-blunt sequence in the ribonucleic acid construct. This is typically embodied as a sequence of single nucleotides, or sequence of nucleotides that are unpaired, located terminally, at the 3'- or 5'-end of said ribonucleic acid construct.

[0070] Such a single-stranded overhang may be a guanosine-rich overhang. That means an overhang that contains a substantial, as defined herein, proportion of guanosine nucleotides.

[0071] The ribonucleic acid construct of the present invention may have said singlestranded overhang at the 5'- or the 3'-end of at least one, meaning at one or at both strands of the double-stranded ribonucleic acid sequence.

[0072] This single-stranded overhang may consist of 3 to 15 nucleosides, preferably 3 nucleosides, preferably containing at least 3 guanosines, more preferably three consecutive guanosines.

[0073] Preferably, the single-stranded overhang at at least one strand of the doublestranded ribonucleic acid sequence has a length of at least 3 nucleosides.

[0074] It is also preferred for the ribonucleic acid construct of the present invention that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 15 nucleosides, more preferably a length of 3 to 10nucleosides, more preferably a length of 3 to 8 nucleosides, more preferably a length of 3 to 5 nucleosides.

[0075] In some embodiments of the ribonucleic acid construct of the present invention, the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least two guanosines.

[0076] In some embodiments of the ribonucleic acid construct of the present invention, the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least three guanosines.

[0077] It is also preferred for the ribonucleic acid construct of the present invention that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises at least three guanosines.

[0078] It is further preferred for the ribonucleic acid construct of the present invention that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three guanosines. More preferably, the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three consecutive guanosines.

[0079] It is also preferred for the ribonucleic acid construct of the present invention that the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises or consists of adenosine-adenosine-guanosine-guanosine- guanosine (AAGGG).

[0080] The single-stranded overhang may also be an overhang, which is self- complementary, for example self-complementary sequences containing internucleotide modifications. Such modifications may be used for securing enhanced 3'-exonuclease stability.

[0081] Double-stranded ribonucleic acid sequence

[0082] In some embodiments of the ribonucleic acid construct of the present invention the double-stranded ribonucleic acid sequence is a blunt-ended ribonucleic acid sequence. This means that the double-stranded ribonucleic acid sequence is a blunt-ended ribonucleic acid sequence apart from the single-stranded overhang at at least one strand of said double-stranded ribonucleic acid sequence, which comprises at least one guanosine. An example of the present invention wherein the double-stranded ribonucleic acid sequence is a blunt-ended ribonucleic acid sequence is that the double stranded ribonucleic acid sequence comprises at the 5'-end of one strand the triphosphate and the single-stranded overhang is at the 3'-end of the second strand such that both strands can comprise at the other ends of both strands a blunt end.

[0083] In the case of the double-stranded ribonucleic acid sequence, the length of the ribonucleic acid sequence is the length of the individual strands.

[0084] It is preferred for the ribonucleic acid construct of the present invention that the double-stranded ribonucleic acid sequence has a length of 8 to 15 ribonucleotides. It is more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 8 to 12 ribonucleotides. It is even more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 10 to 12 ribonucleotides. It is even more preferred for the ribonucleic acid construct of the present invention that the doublestranded ribonucleic acid sequence has a length of 11 ribonucleotides.

[0085] The double-stranded ribonucleic acid sequence may be self-complementary.

[0086] However, the present invention also comprises that in some embodiments, the double-stranded ribonucleic acid sequence may comprise a further overhang apart from the single-stranded overhang at at least one strand of said double-stranded ribonucleic acid sequence, which comprises at least one guanosine. Preferably, that further overhang may be at the strand, which not comprises the single-stranded overhang comprising the at least one guanosine. Preferably, such further overhang may be a single-stranded RNA- overhang or an overhang with a fluorophore, lipophilic substitution, sterols or vitamins.

[0087] For example, the fluorophore used in the context of the present invention may be FAM, FITC, or any Atto Dye.

[0088] Further, a preferred lipophilic substitution used in the context of the present invention may be cholesterol or derivatives thereof; such as cholesterol-C6, cholesterol- Teg or cholesterol-prolinol; oleate or palmitate. Further substitutions may be Trolox, tocopherol-C8, N-acetylgalactosamine (GalNAc C3) or Ibuprofen.

[0089] Cholesterol-C6 has the following chemical structure:

[0090] Cholesterol-Prolinol has the following chemical structure:

[0091] Cholesterol-Teg has the following chemical structure:

[0092] Palmitate has the following chemical structure:chemical structure:

[0096] N-acetylgalactosamine (GalNAc 03) has the following chemical structure:

[0097] For example, the ribonucleic acid construct may comprise a dsRNA or strands of the following sequences as depicted in Table 1 given below. Table 1 also displays the sequences as used in the context of the present invention and as mentioned herein. Each sequence is given in 5'3'-direction (those sequences according to SEQ ID NOs: 1 - 75 are given in the respective sequence listing of the present application with “t” instead of “u” and “21” as “2” due to sequence listing program requirements). The followingabbreviations apply for this Table and are as they are used in the context of the present application: ppp = triphosphate, mA = 2'-O-methyladenosine, mG = 2'-O-methylguanosine, mC = 2'- methylcytosine, mil = 2'-O-methyluridine, fU = 2'-deoxy-2'fluorouridine, fG = 2'- deoxy-2'-fluoroguanosine, fC = 2'-deoxy-2'-fluorocytosine, LC = locked-nucleic acid- cytosine, * = phosphorothioate backbone modification between two nucleotides, dG = 2'- deoxy-guanosine, T = Thymidine and dC = 2'-deoxycytosine:Table 1 :

[0098] It is preferred for the ribonucleic acid construct of the present invention that one or more of its strands comprise(s) or consist(s) of any one of the SEQ ID NOs: 1 to 6, 15 to 48, 58, 59, 61 , 62, 63, 64 or 65 as described herein.

[0099] It is preferred for the ribonucleic acid construct of the present invention that the ribonucleic acid construct is not or does not comprise or does not consist of a hairpinstructure.

[0100] It is further preferred for the single-stranded overhang that said overhang does not comprise a palindromic sequence.

[0101] Linker moiety L

[0102] It is preferred for the ribonucleic acid construct of the present invention that the double-stranded ribonucleic acid sequence is connected with the single-stranded overhang via a linker moiety L.

[0103] The term “linker moiety”, as used herein, means any component of the aforementioned construct whose primary function comprises joining two or more constituent components of the construct together without serving any inherent signalling or pharmacological function perse.

[0104] That linker moiety L may be any linker moiety that provides enough flexibility for the ribonucleic acid construct of the present invention to induce an immune response and / or to induce RIG-l-activity. It may be a flexible linker moiety, which allows the doublestranded ribonucleic acid sequence to be connected with the single-stranded overhang.

[0105] Such a linker moiety L may be a nucleoside-linker or a hydrocarbon-based linker. Preferably, the hydrocarbon-based linker may comprise 3 to 24 main chain carbon atoms, more preferably it may comprise 6 to 18 main chain carbon atoms, more preferably it may comprise 8 to 16 main chain carbon atoms, more preferably it may comprise 10 to 14 main chain carbon atoms and even more preferably it may comprise 12 main chain carbon atoms. When used herein, the term “main chain atoms” refers to the atoms present in the backbone of the linker moiety L. That backbone may be build by carbon-atoms, but in cases, where the carbon-atoms are replaced by O, N or S, those also count to the total number of main chain atoms.

[0106] In some embodiments, the linker moiety L may be a nucleoside-linker. It is preferred when the linker moiety L is a nucleoside-linker that the nucleoside-linker has alength of at least 2 nucleosides. It is also preferred when the linker moiety L is a nucleoside- linker that the nucleoside-linker has a length of 2 to 10 nucleosides. It is further preferred when the linker moiety L is a nucleoside-linker that the nucleoside-linker is an adenosine- linker, preferably with at least 2 adenosines, more preferably with 2 adenosines.

[0107] It is also preferred for the linker moiety L that it is a hydrocarbon-based linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. In the case of Formula I, the carbon-atoms of the (C2-C3)-alkylene as well as the O-atom counts to the total number of main chain atoms.

[0108] More preferably, the linker-moiety L is a polyalkylene-glycol-linker according to Formula IIwherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7. In the case of Formula II, the two carbon-atoms and the O-atom in the bracket of Formula II count to the total number of main chain atoms.

[0109] It is further preferred that the linker moiety L is a polyalkylene-glycol-linker according to Formula II and wherein n is 6. In this case, the number of the main chain atoms is 18.

[0110] Modification(s)

[0111] The ribonucleic acid construct according to the present invention may comprise at least one modification(s). Such at least one modification(s) may be selected from the group consisting of O-methyl-group-modification(s), preferably 2'-O-methyl-group-modification(s), O-methoxyethyl-modification(s), preferably 2'-O-methoxyethyl- modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), phosphoro-thioate-modification(s), locked nucleic acid (LNA)-modification(s), and internucleoside linkage-modification(s), preferably phosphothioate-, thiophosphate-, phosphoroselenate- and selenophosphate-internucleoside linkage-modification(s). Further, such at least one modification(s) may be selected from the group consisting of O- methyl-group-modification(s), preferably 2'-O-methyl-group-modification(s), O- methoxyethyl-modification(s), preferably 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s) or 2'-deoxy-2'-fluoro-group modification(s), phosphoro-thioate-modification(s), 2'-deoxy-group-modification(s), locked nucleic acid (LNA)-modification(s), and internucleoside linkage-modification(s), preferably phosphothioate-, thiophosphate-, phosphoroselenate- and selenophosphateinternucleoside linkage-modification(s). For example, the phosphoro-thioate- modification(s) may be phosphorothioate-backbone-modification(s) between two nucleotides.

[0112] For the at least one modification(s), it has to be considered that the construct of the present invention is a ribonucleic acid (RNA) construct. Each nucleotide in RNA contains a ribose sugar, with carbons numbered T through 5'. A base is attached to the T-position, in general, adenine (A), cytosine (C), guanine (G), or uracil (II). Adenine and guanine are purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3'-position of one ribose and the 5'-position of the next. The unmodified phosphate groups have a negative charge each, making RNA a charged molecule (polyanion). The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.

[0113] The construct of the present invention may be built up of oligonucleotides which may be short, single- or double-stranded RNA molecules, and include antisense oligonucleotides (ASO), RNA interference (RNAi), and aptamer RNAs. As used herein, the term "oligonucleotide" refers to a polynucleotide formed from a plurality of linked nucleoside units. The terms "oligonucleotide" and "polynucleotide" are used synonymously. Such oligonucleotides can be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods including chemical synthesis, in vitro and in vivo transcription. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, pyrophosphate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g. (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages).

[0114] The oligonucleotides of the invention can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. As used herein, the term "modified nucleoside" is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside. In some embodiments, the modified nucleoside is a 2'- substituted ribonucleoside, an arabino-nucleoside or a 2'-deoxy-2'-substituted- arabinoside.

[0115] The term “O-methyl-group-modification(s)”, as used herein, means that the ribonucleic acid construct is modified such that it comprises one or more O-methyl- group(s). Preferably, it may be substituted to comprise a 2'-O-methyl-group. 2'-O- methylation is a common nucleoside modification of RNA, where a methyl group is added to the 2'-hydroxyl of the ribose moiety of a nucleoside, producing a methoxy-group.

[0116] The term “fluoro-group-modification(s)”, as used herein, means that the ribonucleic acid construct is modified such that it comprises one or more fluoro-group(s).

[0117] The term “phosphoro-thioate-modification(s)”, as used herein, means that the ribonucleic acid construct is modified such that it comprises one or more phosphoro- thioate-group(s). Replacing a non-bridging oxygen in the phosphodiester linkage of RNA by sulfur results in phosphoro-thioate-modified RNA. Phosphoro-thioate-modification of mRNA enhances translation efficiency and thus provides a further strategy to improve mRNA functionality.

[0118] A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2'-oxygen and 4'-carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. This structure provides for increased stability against enzymatic degradation. LNA also offers improved specificity and affinity in base-pairing as a monomer or a constituent of an oligonucleotide. LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide.

[0119] As used herein, the term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" includes ribonucleosides or arabinonucleoside in which the hydroxyl group at the 2' position of the pentose moiety is substituted to produce a 2'-substituted or 2'-O- substituted ribonucleoside. Preferably, such substitution is with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group having 6-10carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be substituted, e.g. with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of 2'-O-substituted ribonucleosides or 2'-O-substituted-arabinosides include, without limitation, 2'-O-methylribonucleosides or 2'- O-methylarabinosides and 2'-O-methoxyethylribonucleosides or 2'-O- methoxyethylarabinosides.

[0120] The term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" also includes ribonucleosides or arabinonucleosides in which the 2'-hydroxyl group is replaced with a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an amino or halo group. Examples of such 2'-substituted ribonucleosides or 2'-substituted arabinosides include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl ribonucleosides or arabinosides.

[0121] The term "oligonucleotide" may also include hybrid and chimeric oligonucleotides. A "chimeric oligonucleotide" is an oligonucleotide having more than one type of internucleoside linkage. One preferred example of such a chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region and non-ionic linkages such as alkylphosphonate or alkylphosphonothioate linkages (see e.g. U.S. Pat. NOs. 5,635,377 and 5,366,878).

[0122] A "hybrid oligonucleotide" is an oligonucleotide having more than one type of nucleoside. One preferred example of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-substituted ribonucleotide region, and a deoxyribonucleotide region (see, e.g. U.S. Pat. NOs. 5,652,355, 6,346,614 and 6,143,881).

[0123] RNA oligonucleotides discussed herein include otherwise unmodified RNA as well as RNA which has been modified (e.g. to improve efficacy), and polymers of nucleoside surrogates.

[0124] Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body.

[0125] Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs", they will of course, because of the modification, include molecules which are not RNAs.

[0126] Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g. non-charged mimics of the ribophosphate backbone.

[0127] All nucleic acid sequences listed herein are in the 5'- to 3'-direction unless otherwise indicated.

[0128] A RNA oligonucleotide may contain self-complementary sequences and may form a hairpin, for example with the sequence 5'-GACCTAGCCTAAAACTAGGTC-3' (SEQ ID NO: 7). The self-complementary sequence may be a palindromic sequence. For example, 5'-AAAGATCCGGATCAAAA-3' (SEQ ID NO: 8).

[0129] Transfection reagent

[0130] In some embodiments of the ribonucleic acid construct of the present invention, said construct is capable of penetrating permeabilized cells without the presence of a transfection reagent.

[0131] The term “transfection reagent” as used herein may mean a reagent that is used to introduce naked or purified nucleic acids into eukaryotic cells, for example. These reagents may include cationic lipids, cationic polymers, cationic polypeptides, and any other formulation reagent for nucleic acids that mediates cytoplasmic delivery across the cell membrane.

[0132] Thus, the ribonucleic acid construct of the present invention is preferably able to penetrate permeabilized cells without the presence of a transfection reagent. This means it will be capable of inducing immune-stimulation in tissues or cells that before were challenging to access.

[0133] Medical uses

[0134] In a further aspect, the ribonucleic acid construct of the present invention may be for use as a medicament.

[0135] In a further aspect, the ribonucleic acid construct of the present invention may be for use in a method of treatment or prevention of a disease.

[0136] That disease may be cancer or an infection, preferably a virus infection. Thus, the ribonucleic acid construct of the present invention may be for use in a method of treatment or prevention of a virus or viral infection.

[0137] The terms “treat” and “prevent” as well as words stemming therefrom, like “treatment” or “prevention”, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or moreconditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumour. Also, for purposes herein, “prevention” can encompass delaying the onset of the cancer, or a symptom or condition thereof.

[0138] As used herein, “anti-viral prevention” or “prevention of a virus or viral infection” refers to the treatment of cells, tissues, or organisms in order to trigger preparatory activity that will protect cells from future or ongoing viral infection, typically via activation of the immune system, preferably the innate immune system. Such prevention may include, but is not limited to, intranasal administration of substances onto nasal epithelial tissue.

[0139] Viral infections may include, but are not limited to, infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), hepatitis C, hepatitis B, herpes simplex virus (HSV), HI -AIDS, poliovirus, encephalomyocarditis virus (EMCV) and smallpox virus. Examples of (+) strand RNA viruses which can be targeted for inhibition include, without limitation, picornaviruses, caliciviruses, nodaviruses, coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples of picornaviruses include enterovirus (poliovirus 1), rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus (encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virus O), and parechovirus (human echovirus 22). Examples of caliciviruses include vesiculovirus (swine vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease virus), "Norwalk-like viruses" (Norwalk virus), "Sapporo-like viruses" (Sapporo virus), and "hepatitis E-like viruses" (hepatitis E virus). Betanodavirus (striped jack nervous necrosis virus) is the representative nodavirus. Coronaviruses include coronavirus (avian infections bronchitis virus) and torovirus (Berne virus). Arterivirus (equine arteritis virus) is the representative arteriviridus. Togaviruses include alphavirus (Sindbis virus) and rubivirus (Rubella virus). Finally, the flaviviruses include flavivirus (Yellow fever virus), pestivirus (bovine diarrhea virus), and hepacivirus (hepatitis C virus).

[0140] In case the disease is cancer, the cancer may preferably be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, in some embodiments, the disease may be a haematological tumour or a solid tumour.

[0141] Construct for use in a method of infiltrating immune cells

[0142] In one further aspect, the ribonucleic acid construct of the present invention may be for use in a method of infiltrating immune cells, preferably T cells. In one further aspect, the ribonucleic acid construct of the present invention may be for use in a method of stimulating immune cells. Thus, the ribonucleic acid may have immunostimulatory activity. As used herein, "immunostimulatory activity" refers to the capability of an agent, such as a molecule or a composition, here of the construct of the present invention, to induce an immune response. Thus, the present invention provides a construct capable of immunostimulatory activity, in particular anti-viral responses, in particular type l-IFN- production.

[0143] In some embodiments, the immunostimulatory activity refers to the type I IFN- inducing activity, in particular, IFN-a inducing activity. As used herein, "IFN-a inducing activity" refers to the capability of an agent, such as a molecule or composition, here of the construct of the present invention, to induce IFN-a production from a cell capable of producing IFN-a. Cells capable of producing IFN-a include, but are not limited to, peripheral blood mononuclear cells (PBMC) (e.g. B cells, dendritic cells (myeloid dendritic cells and plasmacytoid dendritic cells), macrophages, monocytes, natural killer cells, granulocytes), endothelial cells, and cell lines.

[0144] Construct for use in a method of turning cold into hot tumours

[0145] In one further aspect, the ribonucleic acid construct of the present invention may be for use in a method of turning cold into hot tumours. The lymphocyte infiltration and IFN-y status may be key factors for effective therapy by defining a “T cell inflamed” phenotype (“hot tumours”). In contrast, lack of T cells infiltrating the tumor characterizes “non-inflamed” or “cold tumours” (in which other immune populations or myeloid cells can however be observed).

[0146] Vaccine

[0147] In a further aspect, the present invention provides the use of the ribonucleic acid construct according to the present invention as a vaccine. Preferably, the vaccine may further comprise an mRNA-sequence.

[0148] Use as immune adjuvant

[0149] In a further aspect, the present invention provides the use of the ribonucleic acid construct according to the present invention as an immune adjuvant.

[0150] The term “immune adjuvant” as used herein means a substance that increases or modulates the immune response to a vaccine. Adjuvants are often used to modify oraugment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus provide increased immunity to a particular disease. Some adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. The ribonucleic acid construct of the present invention is able to serve as such an immune adjuvant.

[0151] Use of the ribonucleic acid construct in the manufacture of a medicament

[0152] In a further aspect, the present invention provides the use of the ribonucleic acid construct according to the present invention in the manufacture of a medicament for the treatment or prevention of a disease. The disease may be cancer or an infection, preferably a virus infection. In case the disease is cancer, the cancer is preferably selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, for example, the disease may be a haematological tumour or a solid tumour.

[0153] Pharmaceutical composition

[0154] The present invention further provides a pharmaceutical composition comprising the ribonucleic acid construct according to the present invention. That composition may further comprise one or more components, such as a pharmaceutically acceptable carrier, an excipient or a delivery agent.

[0155] In some embodiments, the delivery agent is a complexation agent which forms a complex with the oligonucleotide or the precursor thereof and facilitates the delivery of the oligonucleotide into cells. In some embodiments, the complexation agent is a polymer, preferably a cationic polymer. In a preferred embodiment, the complexation agent is a cationic lipid. In another preferred embodiment, the complexation agent is polyethylenimine (PEI) (K. Wu et al., Brain Research 1008(2):284-287 (May 22, 2004); B. Urban-Klein et al. Gene Therapy 12(5):461-466 (2005)). Additional examples ofcomplexation agent include, but are not limited to, collagen derivatives (Y. Minakuchi et al. Nucleic Acids Research 32(13):e109 (2004)), and biodegradable microspheres such as liposomes (M. Sioud, D. Sorensen, Biochem Biophys Res Commun 312(4):1220-1225 (2003); PY Chien et al. Cancer Gene Therapy 12(3):321-328 (2005)), virosomes (J de Jonge et al. Gene Therapy, 13:400-411 (2006)), SNALPs (JJ Rossi, Gene Therapy 13:583-584 (2006)), ISCOMATRIXo (CSL Limited) (I.D. Davis et al. PNAS 101 (29): 10697- 10702 (July 20, 2004); MJ Pearse, D. Drane, Adv Drug Deliv Rev 57(3):465-474 (Jan 10, 2005)), and poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres (A. Khan et al. J Drug Target 12(6):393-404 (2004)).

[0156] Polyethylenimine (PEI) can be linear or branched. In a preferred embodiment, PEI is in vivo-jetPEI which is a linear PEI developed by PolyPlus-transfection for effective and reproducible delivery of anionic oligonucleotides with low toxicity in vivo. The preferred in vivo routes of administration include, but are not limited to, intravenous, intracerebral and intraperitoneal routes.

[0157] SNALPs stands for Stable-Nucleic-Acid-Lipid Particles and contain a lipid bilayer comprised of a mixture of cationic and fusogenic lipid coated with diffusible polyethylene glycol (PEG). The SNALPs are in the 120 nanometer diameter size range, protect the enclosed nucleic acid from serum nucleases and allow cellular endosomal uptake and subsequent cytoplasmic release of the nucleic acid.

[0158] ISCOMATRIXo is made from saponin, cholesterol and phospholipids under defined conditions and forms cage like structures typically 40 nm in diameter. ISCOMATRIXo has the duel capability of facilitating cargo (e.g. antigen) delivery and stimulating the immune system, both the cellular and humoral immune response.

[0159] The pharmaceutical composition of the invention may further comprise another agent such as an agent that stabilizes the oligonucleotide, in particular, RNA oligonucleotide, e.g. a protein that complexes with the oligonucleotide agent to form an iRNP. Still other agents include chelators, e.g. EDTA (e.g. to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g. a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0160] A formulated composition can assume a variety of states. In some examples, the pharmaceutical composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g. less than 80, 50, 30, 20, or 10 % water). In another example, the oligonucleotide agent is in an aqueous phase, e.g. in a solution that includes water, this form being the preferred form for administration via inhalation.

[0161] The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g. a liposome (particularly for the aqueous phase), or a particle (e.g. a microparticle as can be appropriate for a crystalline composition). Generally, theoligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.

[0162] In some embodiments, it is preferred that the pharmaceutical composition of the present invention does not comprise any transfection reagent(s) or complex(es) or composition(s) thereof. In some embodiments, administration routes may include administration via intranasal spray.

[0163] The pharmaceutical composition of the invention can be used to generate a large amount of type I IFN, in particular, IFN-a, I FN-p, IL-18 and / or I L-1 p, in vitro and / or in vivo. The type I IFN, in particular, IFN-a, IFN-p, IL-18 and / or I L-1 p, can be generated at high quantities from different cellular sources, including both immune and non-immune cells, from different species of vertebrates.

[0164] Construct for use in a method of inducing an immune response

[0165] The present application also provides the ribonucleic acid construct according to the present invention for use in a method of inducing an immune response.

[0166] The ribonucleic acid construct may be an antigen recognizing construct or may be part of an antigen recognizing construct or is comprised in an antigen recognizing construct.

[0167] Further, the antigen recognizing construct is an antibody, or fragment thereof, or a T cell receptor (TCR), or fragment thereof, or a chimeric antigen receptor (CAR), or a fragment thereof.

[0168] The term “antibody” in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or a paratope. Such molecules are also referred to as "antigen binding fragments" of immunoglobulin molecules.

[0169] The term “T cell receptor” in general refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, e. g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to an MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and p chains (also known as TCRa and TCRp, respectively), or y and 8 chains (also known as TCRy and TCR8, respectively).

[0170] Chimeric antigen receptors (CARs) - also known as chimeric immuno-receptors, chimeric T cell receptors or artificial T cell receptors - are receptor proteins that have beenengineered to give T cells the new ability to target a specific antigen. The receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor. CAR T cell therapy uses T cells engineered with CARs to treat cancer. The premise of CAR T immunotherapy is to modify T cells to recognize cancer cells in order to more effectively target and destroy them.

[0171] Method of treatment

[0172] The present application provides in a further aspect a method of treating a disease, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to the present invention to a subject in need thereof.

[0173] In some embodiments, the disease may be cancer or an infection, preferably a virus infection.

[0174] In case when the disease is cancer, the cancer may be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, the disease may also be a haematological tumour or a solid tumour.

[0175] In some embodiments of the method of treating according to the present invention, the ribonucleic acid construct may be administered in a multimerized form. This means that the ribonucleic acid construct forms a multimer such that at least two, e.g. three or four of such complexes are present.

[0176] For said method of treating according to the present invention, the ribonucleic acid construct may be administered by any means known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular, rectal, vaginal, and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical composition may be administered by intravenous or intra-parenteral infusion or injection. The pharmaceutical composition of the present invention may also be administered intra- parenchymally, intrathecally, and / or by stereotactic injection. It is preferred for the method of treating according to the present invention that the therapeutically effective amount of the ribonucleic acid construct may be administered to the subject in need thereof viaparenteral, subcutaneous, intramuscular, intravenous, oral, intratracheal, inhalativ or intranasal route(s).

[0177] For oral administration, the ribonucleic acid construct may be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

[0178] Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0179] Capsules for oral use may include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

[0180] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the ribonucleic acid construct of the present invention may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the present invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0181] The therapeutically effective amount of the ribonucleic acid construct may also be administered as encapsulated formulations to protect the ribonucleic acid construct against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 ; PCT publication WO 91 / 06309; and European patent publication EP-A-43075.

[0182] A suitable dose or therapeutically effective amount of the ribonucleic acid construct may be in the range of 0.001 to 500 milligrams per kilogram body weight of the recipient per day (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 100 milligrams per kilogram, about 1 milligrams per kilogram to about 75 milligrams per kilogram, about 10 micrograms per kilogram to about 50 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

[0183] The step of administering a therapeutically effective amount of the ribonucleic acid construct may be once per day, or the construct may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the construct contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g. using a conventional sustained release formulation which provides sustained release of the oligonucleotide agent over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0184] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the infection or disease / disorder, previous treatments, the general health and / or age of the subject, and other diseases / disorders present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual ribonucleic acid construct according to the present invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model.

[0185] Toxicity and therapeutic efficacy of the ribonucleic acid construct and the pharmaceutical composition comprising said construct of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Ribonucleic acid agents that exhibit high therapeutic indices are preferred.

[0186] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosages of the construct or pharmaceutical compositions comprising the construct of the present invention are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any ribonucleic acid agent used in the method of treating of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the ribonucleic acid agent that includes the IC50 (i.e., the concentration of the test ribonucleic acid agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0187] The administering physician can adjust the amount and timing of the administration of the ribonucleic acid construct of the present invention on the basis of results observed using standard measures of efficacy known in the art or described herein.

[0188] Method of inducing an immune response

[0189] In a further aspect, the present invention relates to a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to the present invention to a subject in need thereof. In some embodiments, the immune response may be induced by activating retinoic acidinducible gene I (RIG-I). In some embodiments, the ribonucleic acid construct is administered in a multimerized form as described above.

[0190] For the method of inducing an immune response of the present invention, it is preferred that the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof via parenteral, subcutaneous, intramuscular, intravenous, oral, intratracheal, inhalativ or intranasal route(s). The definitions given above with regard to these administration routes also apply to the method of inducing an immune response. The embodiments for the method of treating according to the present invention also apply here for the method of inducing an immune response.

[0191] Kit

[0192] In a further aspect, the present invention is directed to a kit for use in medicine comprising the ribonucleic acid construct according to the present invention.

[0193] It is preferred that the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

[0194] It is preferred that - in case the disease is cancer - the cancer may be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, headand neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof. However, the disease may also be a haematological tumour or a solid tumour.

[0195] The invention will be further illustrated by the following, non-limiting items:1. A ribonucleic acid construct capable of inducing an immune response, comprising- a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of at least one strand of said sequence, and- a single-stranded overhang at at least one strand of said sequence, wherein the singlestranded overhang comprises at least one guanosine.2. The ribonucleic acid construct of item 1 , wherein the ribonucleic acid construct is capable of inducing an anti-viral immune response.3. The ribonucleic acid construct according to item 1 or 2, wherein the ribonucleic acid construct is capable of inducing IFN production, preferably of inducing type I IFN production.4. The ribonucleic acid construct according to any one of the preceding items, wherein the ribonucleic acid construct is capable of activating retinoic acid-inducible gene I (RIG-I).5. The ribonucleic acid construct according to any one of the preceding items, wherein the ribonucleic acid construct is capable of binding the receptor of retinoic acid-inducible gene I (RIG-I).6. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence has a length of 8 to 15 ribonucleotides.7. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence has a length of 8 to 12 ribonucleotides.8. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence has a length of 10 to 12 ribonucleotides.9. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence has a length of 11 ribonucleotides.10. The ribonucleic acid construct according to any one of the preceding items, wherein the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which also comprises the single-stranded overhang.11. The ribonucleic acid construct according to any one of items 1 to 9, wherein the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which not comprises the single-stranded overhang.12. The ribonucleic acid construct according to any one of items 1 to 9, wherein the doublestranded ribonucleic acid sequence comprises a triphosphate at the 5'-end of both strands.13. The ribonucleic acid construct according to any one of the preceding items, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of at least 3 nucleosides.14. The ribonucleic acid construct of item 13, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 15 nucleosides.15. The ribonucleic acid construct of item 13 or 14, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 10 nucleosides.16. The ribonucleic acid construct of any one of items 13 to 15, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 8 nucleosides.17. The ribonucleic acid construct of any one of items 13 to 16, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides.18. The ribonucleic acid construct of any one of items 13 to 17, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least two guanosines.19. The ribonucleic acid construct of any one of items 13 to 18, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least three guanosines.20. The ribonucleic acid construct of any one of items 13 to 19, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises at least three guanosines.21. The ribonucleic acid construct of item 20, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three guanosines.22. The ribonucleic acid construct of item 21 , wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three consecutive guanosines, preferably wherein the singlestranded overhang has the sequence guanosine-guanosine-guanosine (GGG).23. The ribonucleic acid construct of any one of items 13 to 22, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises or consists of adenosine-adenosine-guanosine-guanosine-guanosine (AAGGG), and / or wherein the single-stranded overhang does not comprise or consist of a palindromic sequence.24. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence is a blunt-ended ribonucleic acid sequence.25. The ribonucleic acid construct according to any one of items 1 to 23, wherein the double-stranded ribonucleic acid sequence comprises a further overhang, preferably a further overhang at the strand not comprising the single-stranded overhang comprising at least one guanosine.26. The ribonucleic acid construct according to item 25, wherein the further overhang is a single-stranded RNA-overhang or an overhang with a fluorophore or a lipophilic substitution. l. The ribonucleic acid construct according to any one of the preceding items, wherein the double-stranded ribonucleic acid sequence is connected with the single-stranded overhang via a linker moiety L.28. The ribonucleic acid construct according to item 27, wherein the linker moiety L is a nucleoside-linker or a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms.29. The ribonucleic acid construct according to item 27, wherein the linker moiety L is a nucleoside-linker.30. The ribonucleic acid construct according to item 27 or 29, wherein the linker moiety L is a nucleoside-linker having a length of at least 2 nucleosides.31. The ribonucleic acid construct according to any one of items 27, 29 or 30, wherein the linker moiety L is a nucleoside-linker having a length of 2 to 10 nucleosides.32. The ribonucleic acid construct according to any one of items 27, 29, 30 or 31 , wherein the nucleoside-linker is an adenosine-linker, preferably with at least 2 adenosines, more preferably with 2 adenosines.33. The ribonucleic acid construct according to item 27 or 28, wherein the linker moiety L is a hydrocarbon-based linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.34. The ribonucleic acid construct according to any one of items 27, 28, or 33, wherein the linker-moiety L is a polyalkylene-glycol-linker according to Formula IIwherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.35. The ribonucleic acid construct according to item 34, wherein the linker moiety is a polyalkylene-glycol-linker according to Formula II and wherein n is 6.36. The ribonucleic acid construct according to any one of the preceding items, wherein the ribonucleic acid construct comprises at least one modification(s).37. The ribonucleic acid construct according to item 36, wherein the at least one modification(s) is / are selected from the group consisting of O-methyl-group- modification(s), preferably 2'-O-methyl-group-modification(s), O-methoxyethyl- modification(s), preferably 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s) or 2'-deoxy-2'-fluoro-group modification(s), phosphoro-thioate-modification(s), 2'-deoxy-group-modification(s), locked nucleic acid (LNA)-modification(s), and internucleoside linkage-modification(s), preferably phosphothioate-, thiophosphate-, phosphoroselenate- and selenophosphateinternucleoside linkage-modification(s).38. The ribonucleic acid construct according to any one of the preceding items, wherein one or more strand(s) of the ribonucleic acid construct comprise(s) or consist(s) of any one of the SEQ I D NOs: 1 to 6, 15 to 48, 58, 59, 61 , 62, 63, 64 or 65.39. The ribonucleic acid construct according to any one of the preceding items, wherein the ribonucleic acid construct is not or does not comprise or does not consist of a hairpinstructure.40. The ribonucleic acid construct according to any one of the preceding items, wherein the ribonucleic acid construct is capable of penetrating permeabilized cells without the presence of a transfection reagent.41 . The ribonucleic acid construct according to any one of the preceding items for use as a medicament.42. The ribonucleic acid construct according to any one of items 1 to 40 for use in a method of treatment or prevention of a disease.43. The ribonucleic acid construct for use according to item 42, wherein the disease is cancer or an infection, preferably a virus infection.44. The ribonucleic acid construct for use according to item 42 or 43, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.45. The ribonucleic acid construct for use according to item 42 or 43, wherein the disease is a haematological tumour or a solid tumour.46. The ribonucleic acid construct according to any one of items 1 to 40 for use in a method of infiltrating immune cells, preferably T cells.47. The ribonucleic acid construct according to any one of items 1 to 40 for use in a method of turning cold into hot tumours.48. Use of the ribonucleic acid construct according to any one of items 1 to 40 as a vaccine, preferably wherein the vaccine further comprises an mRNA-sequence.49. Use of the ribonucleic acid construct according to any one of items 1 to 40 as an immune adjuvant.50. Use of the ribonucleic acid construct according to any one of items 1 to 40 in the manufacture of a medicament for the treatment or prevention of a disease.51 . The use according to item 50, wherein the disease is cancer or an infection, preferably a virus infection.52. The use according to item 50 or 51 , wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.53. The use according to item 50 or 51 , wherein the disease is a haematological tumour or a solid tumour.54. A pharmaceutical composition comprising the ribonucleic acid construct according to any one of items 1 to 40.55. The ribonucleic acid construct according to any one of items 1 to 40 for use in a method of inducing an immune response.56. The ribonucleic acid construct according to any one of items 1 to 40, wherein the ribonucleic acid construct is an antigen recognizing construct or part of an antigen recognizing construct or is comprised in an antigen recognizing construct.57. The ribonucleic acid construct according to item 56, wherein the antigen recognizing construct is an antibody, or fragment thereof, or a T cell receptor (TCR), or fragment thereof, or a chimeric antigen receptor (CAR), or a fragment thereof.58. A method of treating a disease, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to any one of items 1 to 40 to a subject in need thereof.59. The method of treating according to item 58, wherein the disease is cancer or an infection, preferably a virus infection.60. The method of treating according to item 58, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.61. The method of treating according to item 58, wherein the disease is a haematological tumour or a solid tumour.62. The method of treating according to any one of items 58 to 61 , wherein the ribonucleic acid construct is administered in a multimerized form.63. The method of treating according to any one of items 58 to 62, wherein the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral or intranasal.64. A method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to any one of items 1 to 40 to a subject in need thereof.65. The method according to item 64, wherein the immune response is induced by activating retinoic acid-inducible gene I (RIG- 1).66. The method according to item 64 or 65, wherein the ribonucleic acid construct is administered in a multimerized form.67. The method according to any one of items 64, 65 or 66, wherein the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intravenous, oral or intranasal.68. A kit for use in medicine comprising the ribonucleic acid construct according to any one of items 1 to 40.69. The kit according to item 68, wherein the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

[0196] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0197] As used herein, “a” and “an” refer to not only a single individual, but also a group or species of entities unless otherwise noted.

[0198] All terms used herein bear the meanings that are established in the art, unless otherwise noted. Techniques disclosed herein can be performed by a person skilled in the art following the same present description and / or established protocols such as those disclosed in Molecular cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbour Laboratory, New York), Current Protocols in Molecular Biology (Ausubel et al., 2007, John Wiley & Sons, New York), and Current Protocols in Immunology (Coligan et al., 2007, John Wiley & Sons, New York).

[0199] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0200] As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an", as used herein, refer to "one or more" of the enumerated components. The use of the alternative (e.g. "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include", "have", and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0201] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosedherein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein and herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.EXAMPLES

[0202] Materials & methods

[0203] Cell lines

[0204] THP-1 Dual wt (InvivoGen) were cultured in RPMI 1640 (Gibco) in a humidified incubator at 37°C and 5% CO2. Cell culture media were supplemented with 10% fetal calf serum (Gibco), 100 ll / rnl penicillin, and 100 pg / ml streptomycin (Gibco), 1 mM Natriumpyruvat (Gibco) and 0.1 mM MEM non-essential amino acids (Gibco). All cell lines were verified to be mycoplasma negative by using a mycoplasma-specific PCR approach at regular intervals.

[0205] Lipofection based transfection

[0206] THP-1 Dual wt (InvivoGen) were seeded with 80.000 cells / well in a 96-well plate in 100 pL RPMI medium. Cells were transfected with the indicated concentration of RNA using Lipofectamine 2000 following the manufacturer’s instruction. Briefly, the oligonucleotides and Lipofectamine 2000 dilutions were prepared in OptiMEM (Gibco) and incubated for 5 min. Afterward, the RNA / OptiMEM and Lipofectamin / OptiMEM solutions were mixed in equal volume and incubated for additional 20 min at RT. 25 pL of the mixture was added to each well resulting in the indicated concentration.

[0207] Luciferase Assay

[0208] Luciferase assay was performed 18 h after stimulation. To measure Lucia luciferase production, as surrogate for type I IFN induction, 25 pL of the supernatant was mixed with the same volume of luciferase substrate (1 pg / mL coelenterazine in 100 mM Tris pH 7.4, 300 mM Sodium Ascorbate). Luminescence was immediately measured with the EnVision 2104 Multilaber reader (PerkinElmer).

[0209] Digitonin

[0210] 10 x 106THP1 Dual wt cells were seeded in a 10 cm dish in 20 mL RPMI and treated with 0.1 pL Phorbol-12-myristat-13-acetat (PMA, stock concentration 1 mg / mL, final concentration 5 ng / mL) followed by incubation overnight at 37 °C and 5 % CO2. Cells were washed with prewarmed 1 x PBS. 100 pL cell suspension in RPMI was added per well in a flat-bottom 96-well plate (80 x 103cells / well) and incubated overnight at 37 °C and 5 % CO2. 440 ng RNA was diluted in 200 pL digitonin-containing buffer (50 mM HEPES, pH 7.0, 100 mM KCI, 3 mM MgCI2, 0.1 mM DTT, 85 mM sucrose, 0.2 % BSA, 1 mM ATP, 10 pg / mL digitonin). RPMI media was aspired from the cells and replaced by 50 pL of the RNA solution. Cells were incubated for 30 min at 37 °C and 5 % CO2. Wells wereagain aspired and fresh RPMI (100 pL / well) was added. The luciferase assay was performed 18 h after incubation at 37 °C and 5 % CO2.

[0211] Polyacrylamide gel electrophoresis (PAGE)

[0212] Native PAGE (20%) analysis was carried out on 100 pmol of RNA. The samples were prepared without preheating in 1 x TBE Buffer (89 mM Tris base, 89 mM boric acid, 20 mM EDTA, pH 8) with minor amounts of bromphenol blue and orange G as color markers as well as 50 % glycerol.

[0213] Native PAGE was performed at ambient temperature at 120 V for 60 min. 1 x TBE Buffer was employed as running buffer. Gels were stained with SYBR Safe and visualized by UV illumination using Gel Doc 2000.

[0214] Table 2:

[0215] Human lung adenocarcinoma A549 cells were cultured in Dulbecco's Eagle Medium (DMEM, high glucose, Gibco) in a humidified incubator at 37 °C and 5 % CO2. Cell culture media were supplemented with 10 % fetal bovine serum (Gibco), 100 U / rnL penicillin, and 100 pg / mL streptomycin (Gibco), 1 mM sodium pyruvate (Gibco) and 0.1 mM MEM non-essential amino acids (Gibco).

[0216] For generation ofthe ALI culture, A549 cells were seeded at a density of 1x106cells per mL to the apical side of the transwell in a Corning® Transwell® 6 well plate with an insert diameter of 6.5 mm, 0.4 pm pore size and Polyethylene Terephthalate (PET) membranes. To the basolateral side, 700 pL DMEM was added.

[0217] 72 h after seeding, transwells were transferred to 200 pL STEMCELL™ PneumaCult™-ALI Medium and air lift was performed. The basolateral medium of the ALI cultured cells was refreshed at a two-day interval. 20 days after culturing, ALI cells were transfected with 5 pg of the indicated RNA. Stimulation was repeated after 48 h. 90 h after the primary transfection, cells were washed with DPBS and lysed in 100 pL RLT-Buffer (Qiagen) supplemented with 1 % v / v p-Mercaptoethanol.

[0218] Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany) with slight modifications. 1 volume 70 % ethanol was added and loaded onto a Zymo III CG column (Zymo Research). The column was washed with 350 pL Qiagen RW1 wash buffer with subsequent DNase I on-column digestion (Thermo Fisher Scientific). After45 min of incubation at RT, the column was washed twice with Zymo RNA wash buffer. RNA was eluted with RNAse-free water.

[0219] 400 ng RNA was reverse transcribed with RevertAid Reverse transcriptase (Thermo Fisher Scientific) using random hexamer primers (IDT) according to the manufacturer’s instructions.

[0220] Quantitative real-time PCR (qPCR) was performed on a Quantstudio 5 384-well cycler (Thermo Fisher Scientific) using EvaGreen qPCR Mix II (Bio-Budget Technologies, Germany) as instructed by the manufacturer. The following primers (IDT) were used:ISG15: fw 5’-TGG TGG ACA AAT GCG ACG AA-3’ (SEQ ID NO: 68), rev 5‘-CGT ACC TCG TAG GTG CTG C-3‘ (SEQ ID NO: 69),RSAD2: fw 5‘-CCT GTC CGC TGG AAA GTG TT-3‘ (SEQ ID NO: 70), rev 5'-GAC ACT TCT TTG TGG CGC TC-3’ (SEQ ID NO: 71),IFI44: fw 5’-CCC ATC GOT GAA GGA CAG AA-3’ (SEQ ID NO: 72), rev 5'-ACA TGT ACC ACA CCA GCGTT-3' (SEQ ID NO: 73),GAPDH: fw 5’-CAT CTT CCA GGA GCG AGA TCC C-3’ (SEQ ID NO: 74), rev 5'-TTC ACA CCC ATG ACG AAC AT-3' (SEQ ID NO: 75).Gene upregulation was calculated as 2'AActrelative to the 10mM HEPES control and the house keeping gene GAPDH.

[0221] In vivo

[0222] Mice

[0223] C57BL / 6 mice were obtained from Charles River and kept in-house in the House of Experimental Therapy at the University Hospital Bonn. Mice of both sexes that were 8- 20 weeks old were used throughout the study. All of the mice were housed in individually ventilated cages in groups of up to 5 individuals per cage and maintained on a 12 h light / dark cycle at 22-25 °C temperature and 40-70 % relative humidity under specific- pathogen-free conditions. All of the mice were fed with regular rodent chow and sterilized water ab libitum. All of the procedures used in this study were performed with approval by the responsible animal welfare authority (81-02.04.2022.A047 LANUV NRW).

[0224] In vivo RIG-I stimulation and readout

[0225] Indicated RNA was complexed with in vivo jetPEI (N / P ratio 6) according to the manufacturer’s protocol (Polyplus). Mice were injected i.v. with 12.5 pg RNA.

[0226] Peripheral blood was collected from vena facialis 6 h after injection of the RNA. Blood was directly anticoagulated with Li-Heparin and used for IFN-a sandwich ELISA.Capture antibody RMMA-1 (1 pg / ml, PBL Assay Science, Piscataway NJ, USA); Standard protein: recombinant IFN-a (PBL), primary detection antibody polyclonal rabbit antibody against IFN-a (100 neutralizing units / ml, PBL), secondary detection goat-anti-rabbit IgG- HRP (1 : 5000, Bio-Rad laboratories, Hercules, CA USA).

[0227] Total RNA was extracted from mouse spleen using TRIzol (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.

[0228] 500 ng RNA was treated with DNase I (Thermo Fisher Scientific) and cDNA was transcribed with RevertAid Reverse transcriptase (Thermo Fisher Scientific) according to manufacturer’s instructions.

[0229] Quantitative realtime PCR (qPCR) was performed on a Quantstudio 5 384-well cycler (Thermo Fisher Scientific) using EvaGreen qPCR Mix II (Bio-Budget Technologies, Krefeld, Germany) according to manufacturer’s instructions. The following primers (IDT) were used:Ifl44: fw 5'-GGCACATCTTAAAGGGCCACACTC-3' (SEQ ID NO: 9), rev 5'-CTGTCCTTCAGCAGTGGGTCATG-3' (SEQ ID NO: 10),USP18: fw 5'-CACAACATCGGACAGACGTGTTG-3' (SEQ ID NO: 11), rev 5'-CTTGCTCTCTTCTGCACTCCGAG-3' (SEQ ID NO: 12),TBP1 : fw 5'-TCTACCGTGAATCTTGGCTGTAAA-3' (SEQ ID NO: 13), rev 5'-TTCTCATGATGACTGCAGCAAA-3' (SEQ ID NO: 14).

[0230] TBP1 was used as housekeeping gene.

[0231] Gene upregulation was calculated as 2-AActrelative to the untreated mice and the house keeping gene TBP1 .

[0232] Results

[0233] One construct of the present invention, named “GI2” (SEQ ID NOs: 1 + 2) in the Examples-section, outperforms a recently published RIG-I ligand (RNA-1) (SEQ ID NOs: 54 + 55) in inducing IFN-expression. It was believed up to now that a construct must be at least 20 bp in length and must have certain properties to function as a RIG-I ligand: a 5’- C-sense-strand and a 3’-GGG-antisense-strand. The inventors demonstrated that “GI2” (SEQ ID NOs: 1 + 2), beyond the guanosine-rich overhang, is not sequence dependent in it’s capacity to activate RIG-I (see Figure 4, example 4), providing contrast with RNA-1 (SEQ ID NOs: 54 + 55) and demonstrating adaptability of construction whilst retaining RIG- I activation potency.

[0234] Furthermore, “GI2” (SEQ ID NOs: 1 + 2) can be used to drive IFN-expression in reporter cell lines even in the absence of complexation, nanoparticles, or similartransfection reagents, demonstrating its unique capacity to independently penetrate cells and active RIG-1 following minimal chemical modifications to the aforementioned structure of the construct.

[0235] A key structural element was found to be a single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence of the ribonucleic acid construct of the present invention, wherein the single-stranded overhang comprises at least one guanosine. That single-stranded overhang may be, for example, a singlestranded guanosine-rich overhang with appropriate linker moiety L to connect it to the double-stranded ribonucleic acid sequence of the construct. This goes against prior art that stresses the importance of blunt ended RNA-strands as a necessary motif for RIG-1 activation. Surprisingly, the inventors of the present invention have found that via the same IFN-reporter system, that this guanosine-rich overhang is not only beneficial but is highly important for RIG-1 induced IFN-expression by “GI2” (SEQ ID NOs: 1 + 2) (see Figure 7b, example 7).

[0236] The inventors of the present invention have thus identified a ribonucleic acid construct that is a highly potent activator of RIG-I, thereby inducing IFN expression even in the absence of a transfection reagent.

[0237] Example 1 :

[0238] Example 1 shows that “GI2” (SEQ ID NOs: 1 + 2), a ribonucleic acid construct according to the present invention, is a potent inducer of IFN expression in human monocytes (see Figure 1). THP1-Dual-WT cells were transfected with either “GI2” (see Table 1 , SEQ ID NOs: 1 + 2, dark grey, according to the present invention) or a construct named “Superoligo” (SEQ ID NOs: 49 + 50) (light grey, highly-modified 5’-triphosphate 23 bp dsRNA), in complex with Lipofectamine 2000 and were administered at 3 concentrations (0.01 , 1 and 100 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant, with comparable levels of IFN-expression induced by each construct at all 3 concentrations applied. The results represent two experiments and mean ± SEM is shown.

[0239] Example 2:

[0240] Example 2 shows that the ribonucleic acid construct “GI2” according to the present invention (see Table 1 , SEQ ID NOs: 1 + 2) comprises a superior IFN-inducing effect compared to other commonly used pure ribonucleotide RIG-I ligands (see Figure 2). IFN- expression was quantified via luciferase activity in cultured monocytes following application of “GI2” (SEQ ID NOs: 1 + 2) (dark grey), IVT4 (SEQ ID NO: 51) (light grey, 56 bp long RNA produced via in vitro transcription with a GAAA-loop and a 26 bp longdsRNA part), or GFP2 (SEQ ID NOs: 52 + 53) (white with black border, synthetic triphosphate 24 bp dsRNA). After 18 h exposure, “GI2” (SEQ ID NOs: 1 + 2) outperforms both constructs at all 3 concentrations applied (0.01 , 1 and 100 ng / well) after Lipofectamine 2000 transfection. The results represent two experiments and mean ± SEM is shown.

[0241] Example 3:

[0242] Example 3 shows that “GI2” (SEQ ID NOs: 1 + 2) has a substantially stronger IFN- inducing effect compared to recently published RNA-based RIG-I ligand, RNA-1 (SEQ ID NOs: 54 + 55) (see Figure 3). RNA-1 (SEQ ID NOs: 54 + 55) (light grey) is a pure dsRNA construct comprising at least 20 bp and a sense strand: 5’-C, and antisense strand: 3’- GGG. Constructs of either “GI2” (see Table 1 , SEQ ID NOs: 1 + 2, dark grey) or RNA-1 (SEQ ID NOs: 54 + 55) were applied at 3 concentrations and luciferase intensity was quantified, demonstrating that “GI2” (SEQ ID NOs: 1 + 2) is substantially more powerful in inducing IFN-expression at all 3 concentrations following a 18 h exposure. The results represent two experiments and mean ± SEM is shown.

[0243] Example 4:

[0244] Example 4 shows that the IFN-stimulating capacity of “GI2” (SEQ ID NOs: 1 + 2) is independent of RNA nucleotide sequence (see Figure 4). Three constructs differing in nucleotide sequence of 11 bp dsRNA, but each comprising a 5’-triphosphate, a 3’- guanosine-rich overhang with adenosine linker, were applied to THP1 Dual wt. Each construct (“GI2” in dark grey, SEQ ID NOs: 1 + 2, “GI3” in light grey, SEQ ID NOs: 3 + 4, and “GI4” in white with black border, SEQ ID NOs: 5 + 6) induced comparable levels of IFN-expression, indicating that the ability of the described construct to induce IFN- expression does not depend on the precise sequence of nucleotides as long as key structural elements are present - in this example, a 5’-triphosphate and 3’-guanosine-rich overhang. This demonstrates that “GI2” (SEQ ID NOs: 1 + 2) is a RIG-I ligand that - also with a rather short / minimal length - contains the essential molecular motifs required for RIG-l-mediated IFN responses. The results represent three experiments and mean ± SEM is shown.

[0245] Example 5:

[0246] Example 5 shows that “GI4” (SEQ ID NOs: 5 + 6) has a superior I FN-inducing effect compared to 5’-triphosphorylated hairpin RNA constructs (see Figure 5). Hairpincontaining, blunt-ended RNA is a widely described RIG-I ligand. Two such constructs, BM65 (SEQ ID NO: 56) (light grey) and IVT4 (SEQ ID NO: 51) (white with black border)were applied at 3 concentrations and subsequent IFN-expression was contrasted with “GI4” (dark grey, SEQ ID NOs: 5 + 6), which does not contain a hairpin, at each concentration. “GI4” (SEQ ID NOs: 5 + 6) can be seen to induce substantially greater levels of IFN expression versus both hairpin-containing constructs at all 3 concentrations following 18 h exposure to the constructs. The results represent two experiments and mean ± SEM is shown.

[0247] Example 6:

[0248] Example 6 shows that “GI2” (SEQ ID NOs: 1 + 2), when modified through three 2’- O-Me-modification(s) as shown in the scheme and according to “GI2-3M + GI2-3M-AS” (SEQ ID NOs: 15 + 16), see Figure 6, black, middle column), can penetrate cells and activate RIG-I in the absence of a lipid based transfection reagent. 20 x 106THP1 Dual wt cells were incubated with 0.1 pg Phorbol-12-myristate-13-acetate (PMA, stock 1 pg / pL) in 20 mL RPMI medium overnight at 37°C and 5 % CO2 in a 10 cm diameter dish. The cells were washed twice with PBS, transferred in a 96-well-plate with 8 x 104cells / well in 100 pL RPMI medium and incubated overnight at 37°C and 5 % CO2. The medium was removed and the cells were incubated for 30 minutes with 100 ng / well of the assigned RNA dissolved in 50 pL Digitonin containing buffer (50 mM HEPES, pH 7.0, 100 mM KCI, 3 mM MgCh, 0.1 mM DTT, 85 mM sucrose, 0.2 % BSA, 1 mM ATP, 10 pg / mL Digitonin). The RNA containing solution was removed and 100 pL RPMI medium was added. 18 h after incubation at 37°C and 5 % CO2, the induced IFN-a was significantly higher for the modified “GI2”. The results represent two experiments and mean ± SEM is shown.

[0249] Example 7:

[0250] Example 7 shows that lengthening of the guanosine-rich overhang leads to multimerization without improved IFN-induction capability (see Figure 7). The guanosinerich overhang, which typically comprises 3 consecutive guanosines, of the GI2 construct (SEQ ID NOs: 1 + 2) (see Figure 7, black) was modified to either lack a guanosine-rich overhang (SEQ ID NOs: 57 + 2) (see Figure 7, shaped bar) or else to contain a guanosinerich overhang comprising either 4 (SEQ ID NOs: 58 + 2) (see Figure 7, dark grey) or 6 (SEQ ID NOs: 59 + 2) (see Figure 7, light grey) sequential guanosine nucleotides. This Example shows that all 4 constructs were loaded onto a 15% native gel (see Figure 7A). Constructs containing overhangs longer than 3 guanosine residues in length resulted in the observation of slow-running band in the gel, indicating a higher molecular weight and strongly indicating construct multimerization. This Example also shows that whilst lack of overhang resulted in a near total loss of IFN induction at all 3 concentrations applied, overhangs longer than 3 consecutive guanosine nucleotides in length did not improve, orsubstantially worsen, IFN induction capability at any concentration (see Figure 7B). THP1- Dual-WT cells were transfected with the four constructs in complex with Lipofectamine 2000 and were administered at 3 concentrations (0.01 , 1 and 100 ng / well). The results represent two experiments and mean ± SEM is shown.

[0251] Example 8:

[0252] Example 8 shows that “GI2” (SEQ ID NOs: 1 + 2) injection induces an antiviral response in vivo (see Figure 8). C57BL / 6 mice were intravenously injected with 12.5 pg of an RNA construct complexed with in vivo jetPEI, a transfection reagent. The following constructs were injected: CA21 (SEQ ID NO: 60) (light grey, n = 8 mice, negative control), IVT4 (SEQ ID NO: 51) (dark grey, n = 8 mice), “GI2” unmodified (black, n = 4 mice), and “GI2” modified (SEQ ID NOs: 44 + 42) (shaded grey, n = 4 mice). With Figure 8A this Example shows 6 and 24 hours post-injection, sera were collected from mice and the concentration of IFN-a measured via ELISA. At 6 hours, both versions of “GI2” (SEQ ID NOs: 1 + 2) and IVT4 (SEQ ID NO: 51) elicited a comparable level of IFN-a production, with CA21 -elicited levels measuring substantially lower. By 24 hours, for all 4 constructs, IFN-a levels had fallen to a range comparable to IFN-a levels present in untreated sera. With Figures 8B and 8C this Example shows that interferon-stimulated gene expression was determined in the spleen of mice. Expression was quantified by qPCR relative to murine tbk1 expression.

[0253] Example 9:

[0254] Example 9 shows that GI2-3M (SEQ ID NO: 15), combined with antisense strands bearing different lipophilic groups, ribonucleic acid constructs according to the present invention as shown in Figure 9, are inducers of IFN expression in human monocytes. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0255] The sequences as used in Figure 9 and Example 9 are as given in the following Table 3. GI2-3M + GI2-3M-AS corresponds to SEQ ID NO: 15 + SEQ ID NO: 16, GI2-3M + 5'-Chol-C6 corresponds to SEQ ID NO: 15 + SEQ ID NO: 17, GI2-3M + 5'-Chol-Pro corresponds to SEQ ID NO: 15 + SEQ ID NO: 18, GI2-3M + 5'-Teg-Chol corresponds to SEQ ID NO: 15 + SEQ ID NO: 19, GI2-3M + 5'-Palmitate corresponds to SEQ ID NO: 15 + SEQ ID NO: 20, GI2-3M + 5'-Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 21 , GI2-3M + 5'-Tocopherol-C8 corresponds to SEQ ID NO: 15 + SEQ ID NO: 22, GI2-3M +5'-lbu corresponds to SEQ ID NO: 15 + SEQ ID NO: 23, GI2-3M + 3'-Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 24, GI2-3M + 3'-lbu corresponds to SEQ ID NO: 15 + SEQ ID NO: 25 and GI2-3M + 3'-GalNac corresponds to SEQ ID NO: 15 + SEQ ID NO: 26.

[0256] Table 3:

[0257] Example 10:

[0258] Example 10 shows that transfection of air liquid interface A549 cells with GI2-3M (SEQ ID NO: 15), combined with antisense strands (SEQ ID NOs: 27 to 31) bearing different lipophilic groups, which are ribonucleic acid constructs according to the present invention and as shown in Figure 10, enhance interferon-stimulated genes (RSAD2, IFI44, ISG15). Repetitive stimulation has been performed at day 0 and day 2 with 5 pg of the indicated RNA. At day 4, cells have been harvested and qPCR performed. The results represent two experiments and mean ± SEM is shown.

[0259] The sequences as used in Figure 10 and Example 10 are as given in the following Table 4. GI2-3M + Chol-C6 corresponds to SEQ ID NO: 15 + SEQ ID NO: 27, GI2-3M + Chol-Pro corresponds to SEQ ID NO: 15 + SEQ ID NO: 28, GI2-3M + Chol-Teg corresponds to SEQ ID NO: 15 + SEQ ID NO: 29, GI2-3M + Trolox corresponds to SEQ ID NO: 15 + SEQ ID NO: 30 and GI2-3M + Ibuprofen corresponds to SEQ ID NO: 15 + SEQ ID NO. 31.

[0260] Table 4:

[0261] Example 11 :

[0262] Example 11 shows that different 2'-modified ribonucleic acid constructs according to the present invention as shown in Figure 11 are inducers of IFN expression in human monocytes. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h viadetected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0263] The sequences as used in Figure 11 and Example 11 are as given in the following Table 5. GFP2 11 AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 33, GFP2 11 AA GGG + 3F AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 34, GFP2 11 3F AA GGG + GFP2 11 A corresponds to SEQ ID NO: 35 + SEQ ID NO: 33, GFP2 11 3F AA GGG + 3F AS corresponds to SEQ I D NO: 35 + SEQ ID NO: 34, GFP2 11 AA GGG + SO 3M AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 36, SO 11 3M AA GGG + SO 3M AS corresponds to SEQ ID NO: 61 + SEQ ID NO: 36, SO 11 3M AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 61 + SEQ ID NO: 36, GFP2 11 AA GGG + 3L AS corresponds to SEQ ID NO: 32 + SEQ ID NO: 38, GFP2 11 3L AA GGG + GFP2 11 AS corresponds to SEQ ID NO: 37 + SEQ ID NO: 33 and GFP2 11 3L AA GGG + 3L AS corresponds to SEQ ID NO: 37 + SEQ ID NO: 38.

[0264] Table 5:

[0265] Example 12:

[0266] Example 12 shows different modified ribonucleic acid constructs, according to the present invention and as shown in Figure 12, which are inducers of IFN expression in human monocytes. pppGI2-AA-GGG + GI2-AS corresponds to ppp-SEQ ID NO: 39 + SEQ ID NO: 40, pppGI2-AA-3M + GI2-AS-3M corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 42, pppGI2-AA-3M + GI2-AS-3M_2dT corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 43, pppGI2-mod1 + GI2-AS-3M corresponds to ppp-SEQ ID NO: 44 + SEQ ID NO: 42, pppGI2-3M-7T + GI2-AS-3M2T corresponds to SEQ ID NO: 45 + SEQ ID NO: 43, pppGI2-3M-7T + GI2-AS-3M corresponds to ppp-SEQ ID NO: 45 + SEQ ID NO. 42, pppGI2-AA-3M + GI2-AS-3M2T corresponds to ppp-SEQ ID NO: 41 + SEQ ID NO: 46. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0267] The sequences as used in Figure 12 and Example 12 are as given in the following Table 6:

[0268] Table 6:

[0269] Example 13:

[0270] Example 13 shows highly modified ribonucleic acid constructs, according to the present invention and as shown in Figure 13, which are an inducer of IFN expression in human monocytes. pppGI2-AA-GGG + GI2-AS corresponds to SEQ ID NO: 1 + SEQ ID NO: 2 and pppSO-11-3F-dG + SO-3F-2xDNA-AS corresponds to SEQ ID NO: 47 + SEQ ID NO: 48. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0271] The sequences as used in Figure 13 and Example 13 are as given in the following Table 7.

[0272] Table 7:

[0273] Example 14:

[0274] Example 14 shows ribonucleic acid constructs with different connected GGG- overhangs, according to the present invention and as shown in Figure 14, which are an inducer of IFN expression in human monocytes. “GI2: AA-GGG” corresponds to SEQ ID NO: 1 + SEQ ID NO: 2, “GI2: GGG” corresponds to SEQ ID NO: 62 + SEQ ID NO: 2, “GI2: HEG GGG” corresponds to SEQ ID NO: 62 + SEQ ID NO: 2. THP1-Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0275] The sequences as used in Figure 14 and Example 14 are as given in the following Table 8.

[0276] Table 8:

[0277] Example 15:

[0278] Example 15 shows ribonucleic acid constructs with different G-content in the- overhang of the strand not comprising the 5’-ppp-end, according to the present invention and as shown in Figure 15, which are an inducer of IFN expression in human monocytes. “GI2” corresponds to SEQ ID NO: 1 + SEQ ID NO: 2, “GI2 AS: AA GGG” corresponds to SEQ ID NO: 64 + SEQ ID NO: 65, “GI2 AS: AA GG” corresponds to SEQ ID NO: 64 + SEQ ID NO: 66, “GI2 AS: AA G” corresponds to SEQ ID NO: 64 + SEQ ID NO: 67. THP1- Dual-WT cells were transfected with different RNA constructs in complex with Lipofectamine 2000 and were administered at 4 concentrations (100, 10, 1 and 0.1 ng / well). The level of IFN-production stimulated was analysed after 18 h via detected levels of luciferase activity in the supernatant. The results represent two experiments and mean ± SEM is shown.

[0279] The sequences as used in Figure 15 and Example 15 are as given in the following Table 9.

[0280] Table 9:

Claims

CLAIMS:

1. A ribonucleic acid construct capable of inducing an immune response, comprising- a double-stranded ribonucleic acid sequence with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of at least one strand of said sequence, and- a single-stranded overhang at at least one strand of said sequence, wherein the singlestranded overhang comprises at least one guanosine.

2. The ribonucleic acid construct of claim 1 , wherein the ribonucleic acid construct is capable of inducing an anti-viral immune response.

3. The ribonucleic acid construct according to claim 1 or 2, wherein the ribonucleic acid construct is capable of inducing IFN production.

4. The ribonucleic acid construct according to claim 3, wherein the ribonucleic acid construct is capable of inducing type I IFN production.

5. The ribonucleic acid construct according to any one of the preceding claims, wherein the ribonucleic acid construct is capable of activating retinoic acid-inducible gene I (RIG- I).

6. The ribonucleic acid construct according to any one of the preceding claims, wherein the ribonucleic acid construct is capable of binding the receptor of retinoic acid-inducible gene I (RIG-I).

7. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence has a length of 8 to 15 ribonucleotides.

8. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence has a length of 8 to 12 ribonucleotides.

9. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence has a length of 10 to 12 ribonucleotides.

10. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence has a length of 11 ribonucleotides.

11. The ribonucleic acid construct according to any one of the preceding claims, wherein the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which also comprises the single-stranded overhang.

12. The ribonucleic acid construct according to any one of claims 1 to 10, wherein the at least one triphosphate is at the 5'-end of the strand of the double-stranded ribonucleic acid sequence which not comprises the single-stranded overhang.

13. The ribonucleic acid construct according to any one of claims 1 to 10, wherein the double-stranded ribonucleic acid sequence comprises a triphosphate at the 5'-end of both strands.

14. The ribonucleic acid construct according to any one of the preceding claims, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of at least 3 nucleosides.

15. The ribonucleic acid construct of item 14, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 15 nucleosides.

16. The ribonucleic acid construct of claim 14 or 15, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 10 nucleosides.

17. The ribonucleic acid construct of any one of claims 14 to 16, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 8 nucleosides.

18. The ribonucleic acid construct of any one of claims 14 to 17, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides.

19. The ribonucleic acid construct of any one of the preceding claims, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least two guanosines.

20. The ribonucleic acid construct of any one of the preceding claims, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises at least three guanosines.

21. The ribonucleic acid construct of any one of claims 14 to 20, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises at least three guanosines.

22. The ribonucleic acid construct of claim 21 , wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three guanosines.

23. The ribonucleic acid construct of claim 22, wherein the single-stranded overhang at at least one strand of the double-stranded ribonucleic acid sequence has a length of 3 to 5 nucleosides and comprises three consecutive guanosines.

24. The ribonucleic acid construct of claim 23, wherein the single-stranded overhang has the sequence guanosine-guanosine-guanosine (GGG).

25. The ribonucleic acid construct of any one of claims 14 to 24, wherein the singlestranded overhang at at least one strand of the double-stranded ribonucleic acid sequence comprises or consists of adenosine-adenosine-guanosine-guanosine-guanosine (AAGGG).

26. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence is a blunt-ended ribonucleic acid sequence.

27. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence comprises a further overhang, preferably a further overhang at the strand not comprising the single-stranded overhang comprising at least one guanosine.

28. The ribonucleic acid construct according to claim 27, wherein the further overhang is a single-stranded RNA-overhang or an overhang with a fluorophore or a lipophilic substitution.

29. The ribonucleic acid construct according to any one of the preceding claims, wherein the double-stranded ribonucleic acid sequence is connected with the single-stranded overhang via a linker moiety L.

30. The ribonucleic acid construct according to claim 29, wherein the linker moiety L is a nucleoside-linker or a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms.

31. The ribonucleic acid construct according to claim 29, wherein the linker moiety L is a nucleoside-linker.

32. The ribonucleic acid construct according to claim 29 or 31 , wherein the linker moiety L is a nucleoside-linker having a length of at least 2 nucleosides.

33. The ribonucleic acid construct according to any one of claims 29, 31 or 32, wherein the linker moiety L is a nucleoside-linker having a length of 2 to 10 nucleosides.

34. The ribonucleic acid construct according to any one of claims 29, 31 , 32 or 33, wherein the nucleoside-linker is an adenosine-linker, preferably with at least 2 adenosines, more preferably with 2 adenosines.

35. The ribonucleic acid construct according to claim 29 or 30, wherein the linker moiety L is a hydrocarbon-based linker according to Formula IFormula I, wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

36. The ribonucleic acid construct according to any one of claims 29, 30 or 33, wherein the linker-moiety L is a polyalkylene-glycol-linker according to Formula IIwherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

37. The ribonucleic acid construct according to claim 35, wherein the linker moiety is a polyalkylene-glycol-linker according to Formula II and wherein n is 6.

38. The ribonucleic acid construct according to any one of the preceding claims, wherein the ribonucleic acid construct comprises at least one modification(s).

39. The ribonucleic acid construct according to claim 38, wherein the at least one modification(s) is / are selected from the group consisting of O-methyl-group- modification(s), preferably 2'-O-methyl-group-modification(s), O-methoxyethyl- modification(s), preferably 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s) or 2'-deoxy-2'-fluoro-group modification(s), phosphoro-thioate-modification(s), 2'-deoxy-group-modification(s), locked nucleic acid (LNA)-modification(s), and internucleoside linkage-modification(s), preferably phosphothioate-, thiophosphate-, phosphoroselenate- and selenophosphateinternucleoside linkage-modification(s).

40. The ribonucleic acid construct according to any one of the preceding claims, wherein one or more strand(s) of the ribonucleic acid construct comprise(s) or consist(s) of any one of the SEQ ID NOs: 1 to 6, 15 to 48, 58, 59, 61 , 62, 63, 64 or 65, and / or the ribonucleic acid construct is not or does not comprise or does not consist of a hairpin-structure, and / or wherein the single-stranded overhang does not comprise or does not consist of a palindromic sequence, and / or wherein the ribonucleic acid construct is capable of penetrating permeabilized cells without the presence of a transfection reagent.41 . The ribonucleic acid construct according to any one of the preceding claims for use as a medicament.

42. The ribonucleic acid construct according to any one of claims 1 to 40 for use in a method of treatment or prevention of a disease.

43. The ribonucleic acid construct for use according to claim 42, wherein the disease is cancer or an infection, preferably a virus infection.

44. The ribonucleic acid construct for use according to claim 42 or 43, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.

45. The ribonucleic acid construct for use according to claim 42 or 43, wherein the disease is a haematological tumour or a solid tumour.

46. The ribonucleic acid construct according to any one of claims 1 to 40 for use in a method of infiltrating immune cells, preferably T cells.

47. The ribonucleic acid construct according to any one of claims 1 to 40 for use in a method of turning cold into hot tumours.

48. Use of the ribonucleic acid construct according to any one of claims 1 to 40 as a vaccine, preferably wherein the vaccine further comprises an mRNA-sequence.

49. Use of the ribonucleic acid construct according to any one of claims 1 to 40 as an immune adjuvant.

50. Use of the ribonucleic acid construct according to any one of claims 1 to 40 in the manufacture of a medicament for the treatment or prevention of a disease.51 . The use according to claim 50, wherein the disease is cancer or an infection, preferably a virus infection.

52. The use according to claim 50 or 51 , wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.

53. The use according to claim 50 or 51 , wherein the disease is a haematological tumour or a solid tumour.

54. A pharmaceutical composition comprising the ribonucleic acid construct according to any one of claims 1 to 40.

55. The ribonucleic acid construct according to any one of claims 1 to 40 for use in a method of inducing an immune response.

56. The ribonucleic acid construct according to any one of claims 1 to 40, wherein the ribonucleic acid construct is an antigen recognizing construct or part of an antigen recognizing construct or is comprised in an antigen recognizing construct.

57. The ribonucleic acid construct according to claim 56, wherein the antigen recognizing construct is an antibody, or fragment thereof, or a T cell receptor (TCR), or fragment thereof, or a chimeric antigen receptor (CAR), or a fragment thereof.

58. A method of treating a disease, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to any one of claims 1 to 40 to a subject in need thereof.

59. The method of treating according to claim 57, wherein the disease is cancer or an infection, preferably a virus infection.

60. The method of treating according to claim 58, wherein the disease is cancer and the cancer is selected from the group consisting of medulloblastoma, retinoblastoma,Hodgkin's lymphoma, oral cancer, skin cancer, basalioma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, multiple myeloma, prostate cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, melanoma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, mesothelioma cancer, bladder cancer, anal cancer, chondrosarcoma cancer, osteosarcoma cancer, sarcoma cancer, primitive neuroectodermal cancer (primitive neuroectodermal tumour (PNET)), and combinations thereof.61 . The method of treating according to claim 58, wherein the disease is a haematological tumour or a solid tumour.

62. The method of treating according to any one of claims 58 to 61 , wherein the ribonucleic acid construct is administered in a multimerized form.

63. The method of treating according to any one of claims 58 to 62, wherein the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof parenteral, subcutaneous, intratracheal, intrahalativ, intramuscular, intravenous, oral or intranasal.

64. A method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the ribonucleic acid construct according to any one of claims 1 to 40 to a subject in need thereof.

65. The method according to claim 64, wherein the immune response is induced by activating retinoic acid-inducible gene I (RIG- 1).

66. The method according to claim 64 or 65, wherein the ribonucleic acid construct is administered in a multimerized form.

67. The method according to any one of claims 64, 65 or 66, wherein the therapeutically effective amount of the ribonucleic acid construct is administered to the subject in need thereof parenteral, subcutaneous, intramuscular, intratracheal, intrahalativ, intravenous, oral or intranasal.

68. A kit for use in medicine comprising the ribonucleic acid construct according to any one of claims 1 to 40.

69. The kit according to item 68, wherein the kit is a diagnostic kit for selecting a patient for treatment of a disease, preferably wherein the disease is an infection or cancer.

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