Photocontrollable conjugate, pharmaceutical composition and kit comprising the same, and uses thereof

Abstract

The present invention relates to a light-activatable photocaged conjugate, which, upon activation, is capable of inducing an immune response, wherein said conjugate comprises: (a) at least one double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and (b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the at least one double-stranded oligonucleotide according to item (a). The present invention moreover provides a pharmaceutical composition, a drug delivery system, an adjuvant, a vaccine and kit for use in medicine, each of them comprising a conjugate according to the invention. The present invention also relates to said conjugate for use as a medicament, for use as a vaccine, for use as an adjuvant, 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.

Description

International Patent ApplicationApplicants: Rheinische Friedrich-Wilhelms-Universitat BonnJohann Wolfgang Goethe-Universitat FrankfurtOur ref.: RUB18154PCTDate: 27 August 2025PHOTOCONTROLLABLE CONJUGATE, PHARMACEUTICAL COMPOSITION AND KIT COMPRISING THE SAME, AND USES THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to a conjugate comprising: (a) at least one doublestranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and (b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of said at least one double-stranded oligonucleotide according to item (a). The at least one double-stranded oligonucleotide of said conjugate, upon photolytic conversion (i.e. removal of the at least one photoremovable protecting group(s) (PPGs)), can be able to bind to retinoic acidinducible gene I (RIG-I) and to induce activation of the innate immune system. The present invention moreover provides a pharmaceutical composition, a drug delivery system and kit for use in medicine, each of them comprising the conjugate. The present invention also relates to said conjugate for use as a medicament, for use as a vaccine, for use as an immune adjuvant, 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. Described herein is further the use of the conjugate according to the invention in the manufacture of a medicament for the treatment or prevention of a disease, a method of treating a disease and a method of inducing an immune response.BACKGROUND OF THE INVENTION

[0002] The concept of photocages dates back several decades and many molecules with photocontrolled activity have been developed and reported. The basic principle behind photocontrolled I photoresponsive molecules is to mask the biological activity of a given molecule by coupling a photoremovable I photolabile I photocleavable I photoactivatable protecting group ("caging group"; PPG) to a functional group of the molecule so that the molecule loses its activity. By removing the caging group with light irradiation, i.e., using photon energy to initiate a photocleavage reaction ("decaging" / "uncaging"), the activity of the molecule can be restored on demand in aspatiotemporally controlled manner. Due to the orthogonality of light with biological systems and its precise control over the wavelength and irradiation dose, light- controlled traceless activation of molecules is a mild and non-invasive technique that is widely used in biological studies and the biomedical field. The approach has, for example, been successfully applied in targeted cancer therapy using compounds activated by light (Dunkel and lias, 2021), such as in the photochemical control of the activity of a doxorubicin prodrug in deep tumor tissue in vivo (Ibsen et al., 2013), for controlling the activity of other therapeutics (Silva et al., 2019) as well as in the study of biological processes in cells and animals using photocaged biomolecules, including peptides, nucleic acids and proteins, as tools for investigation (Ankenbruck et al., 2017).

[0003] Retinoic-acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) that detects the presence of cytosolic viral RNA and induces a type-l interferon response. The RIG-I receptor is part of the innate immune system and, after activation by its ligand, triggers inter alia intrinsic / mitochondrial apoptosis in tumor cells and the induction of a tumor-specific immune response. In healthy cells, on the other hand, RIG-l-induced apoptosis is prevented by anti-apoptotic factors. RIG-l-based immunological activation can even make therapy-resistant, immunologically inactive ("cold") tumors accessible to therapy by converting them into immunologically active ("hot") tumors. This has made RIG-I ligands highly attractive as immunotherapeutic.

[0004] Artificial RIG-I ligands have been described and are typically embodied by ribonucleic acid (RNA) constructs that contain the requisite molecular motifs to activate RIG-I (for example, see EP 2123757 A1 , EP 2963050 A1 and EP 4015636 A1). Such RIG-I ligands have also been suggested as immunotherapeutic for use in the treatment of tumors and / or viral infections (for example, see EP 2056845 A1 , EP3317410 A1 and EP 3600342 A1). However, systemic non-specific side-effects, such as systemic inflammatory reactions or flu-like symptoms due to uncontrolled overactivation of the immune system, limit the use of RIG-I ligands in immunotherapy. What is not known is how to control activation of RIG-I by its ligands; none of the artificial RIG-I ligands known from the prior art enable control over targeted binding and activation of RIG-I. It would therefore be desirable to find artificial RIG-I ligands whose activity can be controlled, so that undesired systemic side-effects can be significantly reduced or avoided. It would further be desirable to be able to limit the activity to the desired site of application such as, for example, tumor cells.

[0005] mRNA-based vaccines are increasingly recognized as alternative to classical vaccination strategies such as inactivated pathogens or toxins. The two approved mRNA vaccines for SARS-CoV-2 (BioNTech and Moderna) both carry modifications inthe mRNA that cause the immune system to not react to the mRNA itself, but only to the antigen transcribed from the mRNA. These modifications include capping the mRNA and substituting uridine with N1-methyl-pseudouridine. Effective immunization via vaccination typically requires three elements - an antigen (i.e. an immunogenic pathogen-associated protein), adjuvant(s) capable of evoking an innate immune response, and an appropriate delivery vehicle. In the two currently approved mRNA vaccines however no dedicated adjuvants are included in the formulation. Possible components with adjuvant activity such as lipid nanoparticles or unintended RNA products are poorly specified. Activation of RIG-I can serve as an adjuvant for mRNA vaccination (PMID 37428918; EP 24 153 970). However, these formulations may exhibit dosage-limiting side effects due to strong innate immune activation e.g. when injected intratumorally. Additionally, simultaneous RIG-I stimulation can reduce the efficiency of mRNA translation and thus limit antigen availability. It would be desirable to temporally control adjuvant RIG-I activation to ensure optimal amounts of antigen production and to optimize the timing of bystander immune cell activation, as well as to spatially limit RIG-I activation to intended areas of antigen expression to minimize systemic inflammation. Adjuvant(s) having one or more such properties could provide better health outcomes for individuals in need of vaccination in terms of both vaccination success itself and reduction or prevention of unwanted immunotoxic sideeffects.

[0006] In view of the foregoing, there is thus a high need for molecules that allow for temporally and spatially controlled targeted activation of RIG-I thereby enabling to significantly reduce or prevent undesired systemic side-effects and / or to better control immune system activation. A further need is the provision of molecules that can be specifically activated at a desired location, such as tumor cells or the vicinity of tumor cells, and elicit an immune response by activating RIG-I. An objective of the present invention is to address one or more of said needs. A further objective is to overcome the deficiencies, gaps and limitations of the prior art with respect to the spatial and temporal control of RIG-I ligands. Further objectives will become apparent on the basis of the following description, the figures, the non-limiting examples and the claims.SUMMARY OF THE INVENTION

[0007] The inventors have surprisingly found that the conjugate of the present invention provides photocontrollable activation of RIG-I in high-levels and subsequent IFN production. Compared to compounds described in the prior art, the conjugate according to the invention can thus fulfil the hitherto unmet needs described above. In particular, the conjugate of the present invention can act as temporally and spatiallycontrollable RIG-1 ligand, which can be specifically activated at particular desired target locations. Since the conjugate itself is basically comprised of naturally occurring biomolecules and inactive (i.e. unable to activate RIG-1), simple systemic administration without or with significantly reduced adverse side-effects is possible. By the same token, temporal and spatial control over the activation of the immune response is enabled, which can avoid or drastically decrease unwanted activation of the immune system and any adverse effects associated therewith. The conjugate of the present invention can also be part of a vaccine, such as an mRNA-based vaccine, enabling to induce a consistent defined level of innate immune activation during vaccination whilst not negatively influencing vaccine-associated target antigen translation.

[0008] The present invention provides a conjugate comprising: (a) at least one doublestranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and (b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the at least one double-stranded oligonucleotide (a).

[0009] Preferably, said double-stranded oligonucleotide itself (i.e. without any PPG coupled thereto) may be capable of inducing an immune response via activating retinoic-acid inducible gene I (RIG-1). More preferably, the double-stranded oligonucleotide may induce an anti-viral immune response, which may be preferably via induction of interferon expression and release. In other words, in a preferred embodiment, the double-stranded oligonucleotide according to item (a) of the conjugate is an activator of RIG-1 (also referred to as an artificial RIG-1 ligand).

[0010] The double-stranded oligonucleotide may, for example, be a double-stranded ribonucleic acid (RNA). Advantageously, the double-stranded oligonucleotide may, for example, have a length of 8 to 50 ribonucleotides, such as, for example, 10 to 12, preferably 11 , or 20 to 24 ribonucleotides. Double-stranded RNA is generally preferred.

[0011] The double-stranded oligonucleotide may further, for example, advantageously comprise at least one triphosphate at the 5'-end of the first strand or the second strand, preferably at the 5'-end of the first strand. However, the at least one triphosphate can also be at the 5'-end of the second strand or the double-stranded oligonucleotide may comprise a triphosphate at the 5'-end of both stands.

[0012] It may further be preferred that the first and second strand of the double-stranded oligonucleotide form a blunt-end at the 5'-end of the first strand. In a generallypreferred embodiment, the double-stranded oligonucleotide may be a double-stranded RNA without an overhang; the length may, for example, be 20 to 50 ribonucleotides, such as 20 to 24 ribonucleotides. However, it can also be advantageous when the double-stranded oligonucleotide comprises a single-stranded overhang at at least one of the first or second strand, wherein the single-stranded overhang comprises at least one guanosine. The double-stranded oligonucleotide may, for example, have a length of from 8 to 17 ribonucleotides, at least one triphosphate at the 5'-end of at least the first or second strand, and further comprise a single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a triphosphate, wherein the single-stranded overhang comprises at least one guanosine. Said single-stranded overhang may, for example, have a length of at least 3 nucleotides, such as, for example, 3 to 15, 3 to 10, 3 to 8 or 3 to 5 nucleotides. Said overhang may comprise at least two guanosines, such as 3 or more, preferably 3 consecutive, guanosines, for example, adenosine-adenosine-guanosine-guanosine- guanosine (AAGGG). Said single stranded overhang may further advantageously be connected to the double-stranded oligonucleotide via a linker moiety, such as, for example, a nucleotide-linker or a hydrocarbon-based linker.

[0013] In some preferred embodiments of the conjugate of the present invention, the first strand or the second strand of the double-stranded oligonucleotide, e.g., doublestranded RNA, can comprise at least one modification(s). For instance, the at least one modification(s) may be selected from the group consisting of deoxy- modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl- modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro- diamidate-morpholino-modification(s).

[0014] In one embodiment, the at least one double-stranded oligonucleotide of the conjugate may have a structure according to Formula III, Formula IV or Formula V:RNA1 - BL1 - RNA3 RNA1 RNA3RNA2 RNA4 RNA2 - BL2 -RNA4Formula III Formula IVRNA1 - BL1 - RNA3RNA2 - BL2 - RNA4Formula V whereinRNA1 represents the first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA2 represents the second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the first strand via complementary base pairing;RNA3 represents a third strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA4 represents a fourth strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the third strand via complementary base pairing;BL1 , if present, represents a bivalent linker that covalently bonds the 3'-end of RNA1 to the 3'- or 5'-end of RNA3;BL2, if present, represents a bivalent linker that covalently bonds the 5 -end of RNA2 to the respective 5'- or 3'-end of RNA4; wherein

[0015] RNA1 and RNA2, as well as RNA3 and RNA4, each have no overhang of the 5’- terminal nucleotide residues, and wherein RNA1 and RNA2, as well as RNA3 and RNA4, each can comprise an overhang of the 3’-terminal nucleotide residues of not more than five nucleotides, preferably of not more than four or three nucleotides, more preferably of not more than two nucleotides, even more preferably of not more than one nucleotide, especially no overhang of the 3’-terminal nucleotide residues.

[0016] It will be understood that in a conjugate according to the invention, wherein the at least one double-stranded oligonucleotide has a structure according to Formula III, Formula IV or Formula V, the at least one PPG will be coupled to a ribose at the 2'-O- position or a nucleobase of a nucleotide in RNA1 or RNA2. Coupling of the PPG may at each occurrence independently be effected in that the PPG(s) is / are either directly coupled to the 2'-O-position of a ribose or a nucleobase by a photocleavable bond or via a photolabile or self-immolative linker. Moreover, in such conjugates, at least one of RNA3 and RNA4 may, in addition to RNA1 and / or RNA2, also comprise at least one PPG(s). In other words, the one or more PPG(s) may be present in: (i) one of RNA1 or RNA2; (ii) RNA1 and RNA2; (iii) RNA1 and RNA3 or RNA4; (iv) RNA2 and RNA3 or RNA4; (v) RNA1 , RNA3 and RNA4; (vi) RNA2, RNA3 and RNA4 or (vii) RNA1 , RNA2, RNA3 and RNA4, of the conjugate. If two or more PPGs are present in the conjugate, they may be the same or different at each occurrence.

[0017] It is of course also contemplated for such conjugates that at least one of RNA1 , RNA2, RNA3 and RNA4 can comprise at least one modification(s), preferably at least one modification(s) as indicated above.

[0018] Having regard to the bivalent linker BL1 or BL2, each of them may independent from each other, for example, be a nucleotide-linker, a hydrocarbon-based linker, preferably comprising 3 to 24, such as 6 to 18, main chain carbon atoms, a phosphodiester linker (OPO(OH)O-), and any combination thereof.

[0019] The sequence of RNA1 may be identical to the sequence of RNA3. The sequence of RNA2 may be identical to the sequence of RNA4. The length of each of RNA1 , RNA2, RNA3 and RNA4 may independently from each other, for example, be of from 9 to 40, such as 11 to 30 or 20 to 24, nucleotides; or of from 8 to 11 , preferably 11 , nucleotides. The dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 may have the same or a different length. The dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 may each be blunt-ended double-stranded RNA.

[0020] Further possible modifications of a conjugate comprising a double-stranded oligonucleotide according to Formula III, Formula IV or Formula V will become apparent on the basis of the following description, the figures, the non-limiting examples and the claims.

[0021] In one embodiment, the conjugate of the present invention may comprise a structure according to Formula VI or Formula VII:( - \ dsOFormula VI wherein sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand;L3 represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms; dsO represents a double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and (ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand;Formula VII wherein mRNA is a mRNA-strand;(sN-L3-dsO) represents a unit comprising (a) to (c), wherein(a) sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand;(b) L3 represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms;(c) dsO represents a double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and(ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand; and m is an integer in the range of from 1 to 4. It will be understood that in a conjugate according to the invention comprising Formula VI or Formula VII, the at least one PPG will be coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of dsO. It is of course also contemplated for such conjugates that each of the individual elements sN, L3 and dsO, respectively, can be the same or different at each occurrence; the individual constituents of sN, L3 and dsO can also be individually selected. For instance, a conjugate may comprise at least two units (sN-L3-dsO) with identical elements sN, L3 and dsO or units (sN-L3-dsO) having at least one different element sN, L3 or dsO, such as, for example, units having a different sN. The conjugate may preferably comprise at least two units (sN-L3-dsO), such as two, three or four units (sN-L3-dsO), or preferably four units (sN-L3-dsO).

[0022] The sN or sN of each unit (sN-L3-dsO) is preferably complementary to at least one nucleic acid sequence comprised in the mRNA strand, preferably a non-coding nucleicacid sequence of the mRNA-strand, such as, for example, a 3'-untranslated region (UTR)-nucleic acid sequence, a 5 '-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail.

[0023] L3 or L3 in each unit (sN-L3-dsO) may, for example, be bonded to the 3'-end of the first strand or the 5'-end of the second strand of the respective dsO. L3 can, for example, at each occurrence independently be selected from a hydrocarbon-based linker and a polyalkylene-glycol-linker, such as, for example, a polyalkylene-glycol linker according to Formula I(Formula I), wherein n is an integer in the range of 3 to 8, preferably in the range of 5 to 7.

[0024] The dsO or dsO of each unit (sN-L3-dsO) preferably comprises or consists of a double-stranded (ds) RNA, more preferably dsRNA which, after photo-uncaging (i.e. traceless removal of the one or more PPG(s)), is able to act as RIG-1 ligand (i.e. able to bind to and activate RIG-1). The dsO of each unit (sN-L3-dsO) may, for example, be a blunt-ended dsRNA, but dsO(s) which may further comprise an overhang at the first or second strand, such as for example, a RNA-overhang with a fluorophore or a lipophilic substitution, are also contemplated herein. The length of each dsO may independently from each other, for example, be of from 9 to 40, such as 11 to 30 or 20 to 24, nucleotides; or of from 8 to 11 , preferably 11 , nucleotides. In addition, the presence of at least one triphosphate at at least one strand of said dsO may be advantageous, such as, for example, at least one triphosphate at the 5'-end of the first or second strand of said dsO, e.g., dsRNA. The first and / or second strand of each dsO may further, and independently of each other, comprise at least one modification(s), such as, for example, a modification selected from the group consisting of deoxy- modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl- modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro- diamidate-morpholino-modification(s) and any combination thereof. Said at least one modification(s) may be present at the 3’-end and / or the 5’-end of at least the first or second strand of each dsO.

[0025] A conjugate comprising a structure according to Formula VI is able to fulfil a function as immune adjuvant.

[0026] In order to be able to fulfil a function as a vaccine, it is preferred that the mRNA- strand comprises a coding sequence of a protein, a peptide, or an antigen that, once translated, is able to induce an immune response; or a cancer antigen or a cancer- associated antigen. The mRNA-strand may comprise at least one modification(s), such as, for example, modification(s) that can stabilize RNA against degradation, e.g., nuclease degradation, including, for example, pseudouridine-modification(s), e.g. N1- methyl-pseudouridine-modification(s); methoxyuridine-modification(s), e.g. 5- methoxyuridine-modification(s); methylcytosine-modification(s), e.g. 5-methylcytosine- modification(s); methyl-adenosine-modification(s), e.g. N6-methyl-adenosine- modification(s); and any combination of the foregoing modifications.

[0027] Further possible modifications of the conjugate comprising the structure according to Formula VI or VII will become apparent on the basis of the following description, the figures, the non-limiting examples and the claims.

[0028] In a further aspect, the present invention provides the conjugate according to the present invention as described herein for use as a medicament or for use as a vaccine.

[0029] The present invention additionally provides a pharmaceutical composition and a drug delivery system comprising the conjugate according to the present invention. Provided herein is also the conjugate, pharmaceutical composition and delivery system according to the present invention for use in a method of treatment or prevention of a disease.

[0030] The present invention also provides the conjugate or delivery system according to the present invention for use in a method of infiltrating immune cells, preferably T cells.

[0031] Further provided is the conjugate or delivery system according to the present invention for use in a method of turning cold into hot tumors.

[0032] In another aspect, the present invention provides the conjugate according to the present invention for use as an immune adjuvant, and the use of the conjugate as an immune adjuvant is also provided herein.

[0033] The present disclosure also comprises the use of the conjugate according to the present invention in the manufacture of a medicament for the treatment or prevention of a disease.

[0034] In a further aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the present invention.

[0035] The present invention also provides the conjugate according to the present invention for use in a method of inducing an immune response. The released or uncaged double-stranded oligonucleotide of the conjugate according to the present invention, i.e. without PPG(s), may be an antigen recognizing construct or part of an antigen recognizing construct or may be comprised in an antigen recognizingconstruct. 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. Also provided herein is a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the conjugate according to the present invention to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged double-stranded oligonucleotide as immune stimulant at the target site. Oral or parenteral administration may be used. The immune response may be induced by activating or stimulating the retinoic acid-inducible gene I (RIG-I). The conjugate may have or comprise an immune adjuvant function.

[0036] In a further aspect, the present invention provides the conjugate or drug delivery system according to the invention for use in a method of treatment or prevention of a disease, comprising: administering the conjugate or system to a patient; localising the conjugate or system at the target site, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged doublestranded oligonucleotide as therapeutic agent at the target site. The present disclosure also provides a method of treating a disease, comprising the step of administering a therapeutically effective amount of the conjugate or drug delivery system according to the invention to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged doublestranded oligonucleotide as therapeutic agent at the target site. The disease may be cancer or an infection, preferably a virus infection. The disease may also be a hematological tumor or a solid tumor. The conjugate or drug delivery system may be administered orally or parenterally.

[0037] 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.

[0038] The present invention also provides, in a further aspect, a kit for use in medicine comprising the conjugate or drug delivery system according to the present invention and as described herein. The kit may be a diagnostic kit for selecting a patient for treatment of a disease. Preferably, the disease is an infection or cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The invention will be better understood with reference to the detailed description when considered in connection with the non-limiting examples and the accompanying drawings described below.

[0040] Figure 1 shows a schematic representation of an exemplary embodiment of a conjugate according to the present invention and a general exemplary way of activation of the photocaged conjugate. A. Schematic representation of an exemplary embodiment of a conjugate according to the invention, comprising a blunt-ended, double-stranded oligonucleotide (e.g., dsRNA) having a triphosphate moiety attached to the 5'-end of the first strand of the double-stranded oligonucleotide and a photoremovable protection group (PPG) at the N1 position of the first strand of the double-stranded oligonucleotide (indicated by the triangle). B. Schematic representation of an exemplary nucleoside in the double-stranded oligonucleotide with a PPG at the 2'-O-position of the ribose (indicated by the sphere with flash). The PPG makes the double-stranded oligonucleotide inactive, for example, by hindering binding to its target, e.g. RIG-I, so that it cannot interact with its target, e.g. act as RIG-I ligand. C. Schematic representation of an exemplary nucleoside in the double-stranded oligonucleotide after removal of the PPG by light (hv: light energy), i.e. the uncaged nucleoside having the usual hydroxy (OH) group atthe 2'-position of the ribose. Further shown is the general principle of an uncaging reaction, i.e. the conversion of B to C, which restores the activity of the double-stranded oligonucleotide, so that it can interact with its target, e.g. acting as RIG-I ligand capable of inducing an immune response.

[0041] Figure 2 shows an overview of the synthesis of exemplary precursors for the preparation of conjugates according to embodiments of the present invention. A. Overview of the synthesis of 1-(2-nitrophenyl)ethoxy]chloromethyl as precursor of the known photoremovable protection group 1-(2-nitrophenyl)ethoxy]methyl. B. Overview of the synthesis of a protected precursor guanosine having the photoremovable protection group (PPG) 1-(2-nitrophenyl)ethoxy]methyl coupled to its ribose at the 2'- O-position. This protected nucleoside can be introduced into RNA by known automated routine synthesis of RNA resulting in a photocaged RNA strand. C. Overview of the synthesis of a protected precursor cytidine (top row) and a protected precursor adenosine, respectively, having the PPG 1-(2-nitrophenyl)ethoxy]methyl coupled to ribose at the 2'-O-position. These protected nucleosides can be introduced into RNA by known automated routine synthesis of RNA resulting in a photocaged RNA strand.

[0042] Figure 3 shows a schematic representation of exemplary embodiments of a conjugate according to the present invention, comprising at least one double-stranded oligonucleotide having a structure according to Formula III, Formula IV or Formula V. A. Schematic representation of an exemplary embodiment of a conjugate according tothe invention, comprising a double-stranded oligonucleotide having a structure according to Formula III (top) or Formula IV (bottom). In the shown exemplary embodiment of the conjugate comprising the structure of Formula III, depicted are RNA1 and RNA2 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (left side); RNA3 and RNA4 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (right side); BL1 bonding the 3'-end of RNA1 to the 3'-end of RNA3; triphosphate moieties attached to each 5'-end of RNA1 and RNA3, and a photoremovable protection group (PPG) at the N1 position of each of RNA1 and RNA3 (indicated by the triangle). In the shown exemplary embodiment of the conjugate comprising the structure of FormulaIV, depicted are RNA1 and RNA2 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (left side); RNA3 and RNA4 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (right side); BL2 bonding the 5'-end of RNA2 to the 5'-end of RNA4; triphosphate moieties attached to each 5'-end of RNA1 and RNA3, and a photoremovable protection group (PPG) at the N1 position of each of RNA1 and RNA3 (indicated by the triangle). B. Schematic representation of an exemplary embodiment of a conjugate according to the invention, comprising a double-stranded oligonucleotide having a structure according to Formula V. In the shown exemplary embodiment of the conjugate comprising the structure of Formula V, depicted are RNA1 and RNA2 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (left side); RNA3 and RNA4 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (right side); linker BL1 bonding the 3'-end of RNA1 to the 3'-end of RNA3; linker BL2 bonding the 5'-end of RNA2 to the 5'-end of RNA4; triphosphate moieties attached to each 5'- end of RNA1 and RNA3, and a photoremovable protection group (PPG) at the 2'-O- position of N1 of each of RNA1 and RNA3 (indicated by the triangle). C. Schematic representation of an exemplary embodiment of a conjugate according to the invention, comprising a double-stranded oligonucleotide having a structure according to FormulaV. In the shown exemplary embodiment of the conjugate comprising the structure of Formula V, depicted are RNA1 and RNA2 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (left side); RNA3 and RNA4 forming a blunt-ended, double-stranded oligonucleotide (i.e. dsRNA) via complementary base pairing (right side); linker BL1 bonding the 3'-end of RNA1 to the 5'-end of RNA3; linker BL2 bonding the 5'-end of RNA2 to the 3'-end of RNA4; triphosphate moieties attached to each 5'-end of RNA1 and RNA4, and aphotoremovable protection group (PPG) at the N1 position of each of RNA1 and RNA4 (indicated by the triangle).

[0043] Figure 4 shows a schematic representation of exemplary units (sN-L3-dsO) of a conjugate according to the invention, which comprises a structure of Formula VI. A. Depicted are the following of elements of an exemplary unit (sN-L3-dsO): a singlestranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand (sN; bottom); a linker moiety that connects sN and the double-stranded oligonucleotide (wiggled line); and a blunt-ended dsRNA (i.e. element corresponding to dsO)) formed by a first and second strand of ribonucleic acid via complementary base pairing having a triphosphate moiety attached to the 5'-end of the first strand, and a photoremovable protection group (PPG) at the N1 position of the first strand (indicated by the triangle). B. The elements of the depicted exemplary unit (sN-L3-dsO) are identical to that depicted in item A, except that the exemplified unit has two photoremovable protection groups (PPGs) coupled to nucleotides at positions N6 and N7, in 3' to 5' prime direction, of the second strand (indicated by the triangles).

[0044] The units (sN-L3-dsO) can be for use as immune adjuvant whose spatial and temporal activity is controllable by light.

[0045] Figure 5 shows a schematic representation of an uncaged conjugate of an exemplary embodiment of a conjugate comprising the structure according to Formula VII of the present invention. Depicted in this exemplary embodiment is the structure of a mRNA-strand encoding an immunogen and four uncaged units (sN-L3-dsO) hybridized to the mRNA-strand with each unit comprising blunt-ended dsRNA with a triphosphate moiety attached to the 5 -end of each first strand; each blunt-ended dsRNA being capable of acting as RIG-I ligand to bind RIG-I. Such an embodiment may be for use as a vaccine, with the photocaged dsOs providing the required immune- active adjuvant effect in light-controllable manner.

[0046] Figure 6 shows that the IFN-inducing effect of an RNA-based RIG-I ligand can be photocontrolled by a conjugate according to the present invention. In this figure, “neg Ctrl” indicates a sample containing negative control RNA; “N1-2'-O-photocage” indicates a sample containing a conjugate according to the invention (dsRNA with triphosphate ("PPP") at 5’-end of the first strand and coupled PPG at ribose 2'-O- position of the first nucleotide (N1 position) of the first (sense) strand); and “3p-dsRNA” indicates a positive RIG-I ligand control RNA. Samples were peripheral blood mononuclear cells (PBMCs) transfected with 1.28 ng / mL of sample RNA. Samples indicated with +hv were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection. IFN-a production as indicator for RIG-I activation was measured with ELISA after 18 hours of incubation. A more detailed description ofthe experiment is provided in the Examples section. No IFN-a production was observed for the negative control and the non-irradiated cells with photocaged RNA. Transfection with positive RIG-I ligand control RNA resulted in similar levels of IFN-a production for the non-irradiated and irradiated samples. Irradiation of the cells with photocaged RNA elicited a comparable level of IFN-a production as the positive control. This demonstrates that the photocaged dsRNA acts as RIG-I ligand after irradiation with light and that the 2'-O-photocage at position N1 effectively inactivates (masks) essential molecular motifs needed for RIG-I activation, such as, e.g., RIG-l-mediated type I IFN responses. The results represent three experiments and mean ± SEM is shown.

[0047] Figure 7 shows that photocontrollable activation of RIG-I is possible with conjugates according to the invention, which have at least one PPG coupled to different nucleotides in the second strand (antisense strand), and that an increased masking effect is obtained when two PPGs are present. The graph shows the results of the photocontrolled IFN-inducing effect of the RNA-based RIG-I ligand “3p-dsRNA” having a 2'-O-photocage in the second strand (antisense strand “N3.x”) at the following position(s), counted from the 5'-end of the first strand (i.e. 3' to 5' prime direction of the second strand): nucleotide 6 (N3.6), nucleotide 7 (N3.7), and nucleotides 6 and 7 (N3.6 + N3.7). The tested samples were peripheral blood mononuclear cells (PBMCs) transfected with 800 ng / mL of sample RNA. IFN-a production as indicator for RIG-I activation was measured with ELISA 18 hours after transfection for irradiated samples (indicated +hv) and non-irradiated samples (indicated -hv). A more detailed description of the experiment is provided in the Examples section. No or no relevant level of IFN- a production was observed for the negative control (neg Ctrl, right panel), and the nonirradiated cells with photocaged RNA; “N3.6, -hv” (most left panel in black), “N3.7, -hv” (second panel from left in grey), and “N3.6 + N3.7, -hv” (second panel from right in black), Irradiated samples of the photocaged conjugates according to the invention showed levels of IFN-a production (“N3.6, +hv” (most left panel in grey), “N3.7, +hv” (second panel from left in light grey), and “N3.6 + N3.7, +hv” (second panel from right in grey) comparable to that of cells transfected with RIG-I ligand control RNA 3p- dsRNA (right panel in dark grey). The results represent at least three experiments and mean ± SEM is shown.

[0048] Figure 8 shows that the IFN-stimulating activity of “GFP2-AAGGG” and “GI2- HEG-GGG”, which comprise a double-stranded oligonucleotide with a guanosoine- containing overhang as described herein and are potent inducers of IFN expression, can be suppressed by introducing 2'-O-methyl-group-modifactions at positions 6(N3.6) and 7 (N3.7), in a 3' to 5' prime direction, of the second strand. A. Schematic representation of “GFP2-AAGGG + N3.6N3.7 2’0Me”, a blunt-ended dsRNA having a length of 11 bp, a 5’-triphosphate moiety at the first strand, a 3’-guanosine-rich overhang with adenosine linker at the first strand; positions 6 (N3.6) and 7 (N3.7), in a 3' to 5' prime direction, of the second strand indicated. B. Shown are the results of Lucia luciferase induction by a interferone-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen) transfected with “GFP2-AAGGG”, analysed 18 h after transfection via detected levels of luciferase activity in the supernatant (black bars on the left), in comparison to levels in cells transfected with GFP2-AAGGG having 2'-O-methyl-group-modifications at positions N3.6 and N3.7 of the second strand (light grey bar in the middle) and negative control "neg Ctrl" (dark grey bar on the right). C. Shown are the results of Lucia luciferase induction by a interferone-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen) transfected with “GI2-HEG-GGG”, analysed 18 h after transfection via detected levels of luciferase activity in the supernatant (black bar on the left), in comparison to levels in cells transfected with “GI2-HEG-GGG” having 2'-O-methyl- group-modifications at positions N3.6 and N3.7 of the second strand (light grey bar in the middle) and negative control "neg Ctrl" (dark grey bar on the right). The 2'-O-methyl- group-modifications at positions N3.6 and N3.7 of the second strand, inactivate RIG-I ligand activity of “GFP2-AAGGG” and “GI2-HEG-GGG”. This confirms and provides proof of concept that controlled targeted activation of RIG-I can be achieved with dsRNA having a rather short minimal length and an overhang as described herein in a sequence independent manner.

[0049] Figure 9 shows that the IFN-inducing effect of a RNA-based RIG-I ligand can be photo-controlled by a conjugate according to the present invention, which comprises at least one PPG coupled to a nucleobase. A. Depicted is the structure of an exemplary nucleoside that contains a PPG coupled to the nucleobase. Such a nucleoside can be incorporated in the sequence of RNA and / or DNA by standard routine automated synthesis of oligonucleotides. B. Shown are the results of levels of IFN-production stimulated in cells transfected with 800 ng / mL of "3p-dsRNA" without modification and with photocaged nucleobase at position N3.1 of the second strand; samples indicated with +hv were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 18 h after transfection via IFN-a ELISA in the supernatant. Non-irradiated cells with photocaged RNA showed reduced levels of IFN production, while irradiation of cells with photocaged RNA elicited a comparable level of IFN production as the pure "3p-dsRNA"positive control. C-E. Shown are the results of levels of IFN-production stimulated in cells transfected with 800 ng / mL of "ds GI2 II" without modification and with two or more photocaged nucleobases: C. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; D. photocaged nucleobases at positions N3.2 and N3.5 of the second strand; E. photocaged nucleobases at positions N3.1 and N3.2 and N3.5 of the second strand; samples indicated with +hv were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 18 h after transfection via IFN-a ELISA in the supernatant. Non-irradiated cells with photocaged RNA showed significantly reduced levels of IFN production, while irradiation of cells with photocaged RNA elicited a comparable level of IFN production as the pure "ds GI2 II" positive control. This demonstrates that the photocaged dsRNA acts as RIG-I ligand after activation with light and that a photocage on one nucleobase, e.g. at position N1 , effectively inactivates (masks) essential molecular motifs needed for RIG-I activation, such as, e.g., RIG-l-mediated IFN responses. This inactivation is further enhanced upon the introduction of a second or a second and third photocage on other nucleobases of the ligand. All in all, the results presented in this figure provide general proof of concept that photocaging of a nucleobase masks biological activity of the double-stranded oligonucleotide, and that the activity of the double-stranded oligonucleotide can be restored on demand in a spatiotemporally controlled manner by irradiation with light.

[0050] Figure 10 shows that the immune stimulation of a conjugate according to this invention, consisting of a RNA-based RIG-I ligand hybridized to a complementary mRNA can be photocontrolled by coupling of PPGs to different nucleotides in the second strand (antisense strand) of the complementary RIG-I ligand (CRL). Shown are the results of Lucia luciferase induction by a interferone-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen) transfected with 800 ng / mL (calculated on mRNA amount) of mRNA with or without prior hybridisation to a CRL. This CRL is either not photo-controllable (“CRL”) or coupled with a PPG at position N3.6 and N3.7 of the second strand (“photocage-CRL”); the samples were either irradiated (7 min of irradiation, grey) or not irradiated (0 min of irradiation, black) after transfection, and analysis was performed 18 h after transfection with a Lucia luciferase assay in the supernatant. Cells as well as mRNA alone showed no significant levels of immune stimulation. Non-irradiated cells with “photocage CRL” showed reduced levels of Lucia luciferase induction, comparable to the negative control “cells alone”, while irradiation of cells with mRNA coupled to a photocaged CRL elicited a comparable level of Lucia luciferase induction as the pure "mRNA+CRL" positive control. In summary, the results presented in this figure provide general proofof concept that photocaging of a CRL hybridized to a mRNA masks immune stimulatory properties of the construct, and that the immune stimulation activity of the conjugate can be restored on demand in a spatiotemporally controlled manner by irradiation with light.

[0051] Figure 11 shows that the light control over the IFN-inducing effect of the conjugate according to the present invention, which comprises at least one PPG coupled to the 2'-O-position of a nucleotide or to a nucleobase is independent of the used concentration. A and B. Shown are the results of levels of IFN-production stimulated in cells transfected with the indicated concentrations (x-axis) of "3p-dsRNA" without modification and with: A. photocaged 2'-O-nucleotide at position N3.7 of the second strand; and B. photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand. C, D and E. Shown are the results of levels of IFN-production stimulated in cells transfected with the indicated concentrations (x-axis) of "ds GI2 II" without modification and with two or more photocaged nucleobases: C. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; D. photocaged nucleobases at positions N3.2 and N3.5 of the second strand; and E. photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand. A-E. Samples indicated with +hv were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 18 h after transfection via IFN-a ELISA in the supernatant. In all concentrations, non-irradiated cells with photocaged RNA showed reduced levels of IFN production, comparable to the negative control RNA, while irradiation of cells with photocaged RNA elicited a comparable level of IFN production as the pure "3p-dsRNA" (A and B) or “ds GI2 II" (C to E) positive control. The results demonstrate that the light controllable effect of the photocaged dsRNA ligands is independent of the RNA concentration used for stimulation. This concentration independency is observed for the construct regardless of the number of photocages on the conjugate. All in all, the results presented in this figure provide general proof of concept that photocaging of a RNA ligand according to this invention masks biological activity of the double-stranded oligonucleotide in a concentration independent manner.

[0052] Figure 12 demonstrates the stability of the conjugates described herein. In particular, the results of Figure 12 show that there is no spontaneous light-independent cleavage of the coupled photocage(s) when the conjugates described herein are exposed to cellular environments. As demonstrated, the coupled photocage(s) remain(s) stable and inert in the absence of light, thereby preventing unintended activation or release of the conjugate inside the cells. The results show the IFN- inducing effect measured as the ISG CXCL-10 of a conjugate coupled with photocagemodifications at the 2'-O-position of the nucleotides at position N3.6 and N3.7 of the second strand. A to C. Shown are the results from a conjugate as disclosed herein exposed to cellular environments of different cancerous cell lines: A. HeLa (human ovarial carcinoma); B. A375 (human melanoma), for ease of visualization of ranges of values, y-axis has been broken (i.e. portion of y-axis scale cut out); and C. Hcmel3 (murine melanoma). The cell lines were transfected with 800 ng / mL positive control RNA (3p-dsRNA, black bars), negative control RNA (neg Ctrl, middle grey bars) or RNA with photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand. Samples indicated with +hv (black and light grey bars) were irradiated with lightemitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 72 h after transfection via CXCL-10 ELISA in the supernatant. Nonirradiated cells with photocaged RNA showed reduced levels of IFN production (dark grey bars) with no significant difference to the negative control RNA (middle grey bars), while irradiation of cells with photocaged RNA elicited a comparable level of IFN production (light grey bars) as the pure "3p-dsRNA" positive control (black bars). It could be shown that even after 72 hours of incubation no background activation of the non-irradiated photocage construct is happening within the cells. The irradiated photocage conjugates on the other hand were stable and able to stimulate RIG-I as the positive control. All in all, the results presented in Figure 12 provide general proof of concept that photocaged conjugates as described herein are stable within the cell, meaning, that no light-independent cleavage of the photocage occurs and that they can be activated on demand in a spatiotemporally controlled manner.

[0053] Figure 13 demonstrates the controllability of immune induction and cell death induction in primary human clear cell renal carcinoma cells by light using a conjugate according to the present invention. The results in A. show the IFN-inducing effect measured as the ISG CXCL10 of a conjugate coupled with photocage modifications at the 2'-O-position of the nucleotides at position N3.6 and N3.7 of the second strand. B. shows the results of FACS analysis of an annexin V and 7-AAD staining for apoptotic and dead cells (for ease of visualization of ranges of values, y-axis has been broken). The cells were transfected with 800 ng / mL positive control RNA (3p-dsRNA), negative control RNA (neg Ctrl) or RNA with photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand. Apoptosis inducer staurosporine was used as an apoptosis positive control. Samples indicated with “+hv” were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 72 h after transfection via A. CXCL10 ELISA in the supernatant and B. annexin V / 7-AAD staining and following FACS analysis of the cells. Non-irradiated cells with photocaged RNA showed reduced levels of IFN production and cell death induction with nosignificant difference to the negative control RNA, while irradiation of cells with photocaged RNA elicited a comparable level of IFN production and cell death induction as the pure "3p-dsRNA" positive control. A significant difference between nonirradiated and irradiated conditions of the photocaged construct could be shown in both assays. All in all, the results presented in Figure 13 provide proof of concept that photocaged conjugates as described herein can be used to control the immune induction and the cell death induction in primary human cancer cells.

[0054] Figure 14 shows that the conjugate according to the present invention, which comprises at least one PPG coupled to the 2'-O-position of a nucleotide with a length of 24 basepairs can be activated inside the cell for at least 24 hours after transfection. Cells were transfected with 800 ng / mL of "3p-dsRNA" without modification and with photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand as well as single stranded negative control RNA (neg Ctrl). Following transfection cells where irradiated with light-emitting diodes (365 nm, 2 mW, 10 minutes) after the indicated time (legend on the right). 24 hours after irradiation ISRE reporter activity was measured in the supernatant. For the non-irradiated control, ISRE reporter readout was performed 24 hours after transfection. Shown are the results of Lucia luciferase induction by an interferon-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen). Activation of approximately 75% relative to the conjugate irradiated immediately after transfection was observed when cells with the photocaged conjugate were irradiated following a 4-hour incubation period. Notably, even after 24 hours of incubation prior to irradiation, around 50% activation was still achieved. Even after 72 hours of incubation before irradiation the activity of the conjugate exceeded that of the negative control. All in all, the results presented in this figure demonstrate that the conjugate according to this invention can be activated after different incubation times within the cellular environment and still induce a considerable immune response.

[0055] Figure 15 shows the light control over the IFN-inducing effect of the conjugate according to the present invention, which comprises at least one PPG coupled to the 2'-O-position of a nucleotide on a 11 base-pair long ligand with an overhang of the first strand. Cells were transfected with the indicated concentrations (x-axis) of A. "GFP2- AAGGG" without modification and with photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand and of B. "ds GI2 II" without modification and with photocaged 2'-O-nucleotides at positions N3.6 and N3.7 of the second strand. A and B. Shown are the results of levels of IFN-production, measured as IFNa, and the levels of CXCL10 induction. CXCL10, as an interferon-stimulated gene, is a more sensitiveindicator than IFNa for detecting low IFN stimulation. Samples indicated with “+hv” were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection. Supernatants were collected 18 hours after transfection and IFNa and CXCL10 levels were measured by ELISA. In both ligand systems, non-irradiated cells with photocaged RNA exhibited reduced production of IFNa and CXCL10, comparable to the negative control RNA. In contrast, irradiation of cells transfected with photocaged RNA restored IFNa and CXCL10 levels to those observed with the positive controls "GFP2-AAGGG" (A) or “ds GI2 II" (B). This effect was seen across all tested concentrations. The results demonstrate that the light controllable effect of the photocaged dsRNA ligands is independent of the RNA concentration used for stimulation. Overall, the results presented in this figure in combination with the results seen in Figure 11 provide proof that photocaging of an RNA ligand according to this invention effectively masks biological activity of the double-stranded oligonucleotide, regardless of its concentration, length, and type of location of coupled photocage (nucleobase or 2’-O-position).

[0056] Figure 16 demonstrates the functionality of nucleobase photocaged constructs according to this invention for photocontrolled immune induction in various cancer cell lines. In particular, the results of Figure 16 show that there is no spontaneous light-independent cleavage of the coupled photocage(s) when the conjugates described herein are exposed to cellular environments. As demonstrated, the coupled photocage(s) remain(s) stable and inert in the absence of light, thereby preventing unintended activation or release of the conjugate inside the cells. The results show the IFN-inducing effect measured as the ISG CXCL10 of a conjugate with at least one PPG coupled to a nucleobase of a nucleotide with a length of 11 basepairs and an overhang on the first strand in different cancerous cell lines: A. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; B. photocaged nucleobases at positions N3.2 and N3.5 of the second strand; and C. photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand (for ease of visualization of ranges of values, y-axis has been broken). The cell lines were transfected with 800 ng / mL positive control RNA (ds GI2 II, black, dark grey bars), negative control RNA (neg Ctrl, middle grey bars) or RNA with photocaged nucleobases at the described positions of the second strand. Samples indicated with “+hv” (dark and light grey bars) were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 72 h after transfection via CXCL10 ELISA in the supernatant. Non-irradiated cells with photocaged RNA showed reduced levels of IFN production with no significantdifference to the negative control RNA, while irradiation of cells with photocaged RNA elicited a comparable level of IFN production as the pure "ds GI2 II" positive control. Even after 72 hours of incubation no background activation of the non-irradiated photocage construct was observed within the cells. The irradiated photocage conjugates on the other hand were able to stimulate RIG-1 as potently as the positive control. All in all, the results presented in Figure 16 provide general proof of concept that photocage basecaged conjugates as described herein are stable within the cell, meaning, that no light-independent cleavage of the photocage occurs and that they can be activated on demand in a spatiotemporally controlled manner to induce an immune response in cancer cells.

[0057] Figure 17 demonstrates the functionality of nucleobase photocaged constructs according to this invention for photocontrolled cell death induction in various cancer cell lines. In particular, the results of Figure 17 show that there is a significant difference in cell death induction between the non-irradiated and irradiated conjugates described herein with at least one PPG coupled to a nucleobase of a nucleotide with a length of 11 basepairs and an overhang on the first strand in different cancerous cell lines: A. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; B. photocaged nucleobases at positions N3.2 and N3.5 of the second strand (for ease of visualization of ranges of values, y-axis has been broken); and C. photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand (for ease of visualization of ranges of values, y-axis has been broken). The cell lines were transfected with 800 ng / mL positive control RNA (ds GI2 II, black, dark grey bars), negative control RNA (neg Ctrl, middle grey bars) or RNA with photocaged nucleobases at the described positions of the second strand. Samples indicated with “+hv” (dark and light grey bars) were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection. Analysis was performed 72 hours after transfection via FACS analysis of the annexin V / 7-aminoactinomycin D (7-AAD) stained cells. Non-irradiated cells with photocaged RNA showed reduced levels of cell death induction with no significant difference to the negative control RNA, while irradiation of cells with photocaged RNA elicited a comparable cell death induction as the pure "ds GI2 II" positive control. It was demonstrated that even after 72 hours of incubation, no background activation of the non-irradiated photocage construct occurred within the cells. In contrast, the irradiated photocaged conjugates induced cell death with potency comparable of that of the positive control. All in all, the results presented in Figure 17 provide further proof of concept that photocage basecaged conjugates as described herein are stable within the cell, meaning, that no light-independent cleavage of the photocage occurs and that they can be activated on demand in a spatiotemporally controlled manner to induce cell death in cancer cells.

[0058] Figure 18 shows that the conjugate according to the present invention, which comprises at least one PPG coupled to a nucleobase of a nucleotide with a length of 11 basepairs and an overhang on the first strand can be activated inside the cell for at least 4 hours after transfection. Cells were transfected with 800 ng / mL of "ds GI2 II" without modification and with photocaged nucleobases at positions N3.1 and N3.2 of the second strand; photocaged nucleobases at positions N3.2 and N3.5 of the second strand; and photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand, as well as single stranded negative control RNA (neg Ctrl). Following transfection cells where irradiated with light-emitting diodes (365 nm, 2 mW, 10 minutes) after the indicated (legend at the top) time. 24 hours after irradiation ISRE reporter activity was measured in the supernatant. For the non-irradiated control, ISRE reporter readout was performed 24 hours after transfection. Shown are the results of Lucia luciferase induction by an interferon-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen). Activation of approximately 80% relative to the conjugate irradiated immediately after transfection was observed when cells with the photocaged conjugate were irradiated following a 4-hour incubation period. Notably, even after 24 hours of incubation prior to irradiation, approximately 25% activation was still achieved. All in all, the results presented in this figure demonstrate that the conjugate according to this invention can be activated after different incubation times within the cellular environment and still induce a considerable immune response.

[0059] Figure 19 shows the light control over the IFN-inducing effect of the conjugate according to the present invention, which comprises at least one PPG coupled to the nucleobase on a 11 base-pair long ligand with an overhang of the first strand using standard in vivo transfection reagent in vivo-jetPEI. Shown are the results of the IFN- inducing effect measured as the ISG CXCL10 stimulated in cells transfected with the indicated concentrations (x-axis) of “ds GI2 II” without modification and with A. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; B. photocaged nucleobases at positions N3.2 and N3.5 of the second strand; and C. photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand; as well as single stranded negative control RNA (neg Ctrl). A-C. Samples indicated with “+hv” were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 18 hours after transfection via CXCL10 ELISA in the supernatant. All three photocaged ligands showed reduced levels of CXCL10 production in non-irradiated cells with photocaged RNA, comparable to thenegative control RNA. In contrast, irradiation of cells with photocaged RNA elicited a comparable level of CXCL10 production as the pure “ds GI2 II" positive control across all concentrations. The results demonstrate that the light controllable effect of the photocaged dsRNA ligands can be obtained, using standard in vivo transfection reagent in vivo-jetPEI.

[0060] Figure 20 shows the light control over the IFN-inducing effect of the conjugate according to the present invention, which comprises at least one PPG coupled to the nucleobase on a 11 base-pair long ligand with an overhang of the first strand using standard in vivo transfection reagent Invivofectamine 3.0. Shown are the results of the IFN-inducing effect measured as the ISG CXCL10 stimulated in cells transfected with the indicated concentrations (x-axis) of “ds GI2 II” without modification and with A. photocaged nucleobases at positions N3.1 and N3.2 of the second strand; B. photocaged nucleobases at positions N3.2 and N3.5 of the second strand; and C. photocaged nucleobases at positions N3.1 , N3.2 and N3.5 of the second strand; as well as single stranded negative control RNA (neg Ctrl). A-C. Samples indicated with “+hv” were irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes) directly after transfection, and analysis was performed 18 hours after transfection via CXCL10 ELISA in the supernatant. All three photocaged ligands showed reduced levels of CXCL10 production in non-irradiated cells with photocaged RNA, comparable to the negative control RNA. In contrast, irradiation of cells with photocaged RNA elicited a comparable level of CXCL10 production as the pure “ds GI2 II" positive control across all concentrations. The results demonstrate that the light controllable effect of the photocaged dsRNA ligands can be obtained, using standard in vivo transfection reagent Invivofectamine 3.0.

[0061] Figure 21 shows that coupling of an additional fluorophore label, such as Cyanine 3 (Cy3) fluorescent dye, does not negatively affect immune stimulation activity of a conjugate according to the present invention, e.g. a conjugate comprising at least one PPG coupled to a nucleobase, an overhang at the first strand and a length of 11 base-pairs. Cells were transfected with the indicated concentrations (x-axis) of “ds GI2 II” without modification and with photocaged nucleobases at positions N3.2 and N3.5 of the second strand without or with an additional Cy3 label at the 5’-end of the second strand. Following transfection samples indicated with “+hv” were irradiated with lightemitting diodes (365 nm, 2 mW, 10 minutes). 24 hours after transfection, ISRE reporter activity was measured in the supernatant und cell fluorescence was imaged with fluorescence microscopy. A. Shown are the results of Lucia luciferase induction by an interferon-stimulated-response element (ISRE) reporter stimulated in THP1 dual™ cells (InvivoGen). B. Shown are images of the cells using bright field light (top row) ora fluorescence channel for Cy3 fluorescence (middle row,ex=555 nm) and the merged image of both (bottom row). C. Shown is the quantification of the mean fluorescence signal in the Cy3 images from B. Photocaged conjugates both, without or with Cy3 label, showed reduced levels of immune stimulation in non-irradiated cells compared to irradiated cells transfected with the same constructs. In contrast, irradiation of cells with photocaged RNA elicited a comparable level of immune stimulation as the pure “ds GI2 II" positive control across all concentrations. The fluorescence images and their signal quantification demonstrate an equal distribution of the Cy3-labeled photocaged construct in the cells, regardless of irradiation. Thus, the photocaged ligand is indeed inactive inside the cellular environment in the absence of light. The results demonstrate that fluorescent labels according to this invention can be added to the conjugates according to this invention without an influence on the functionality of the conjugate.DETAILED DESCRIPTION OF THE INVENTION

[0062] As stated before, the invention described herein concerns a conjugate comprising: (a) at least one double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and (b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the at least one double-stranded oligonucleotide (a).

[0063] None of the structures known from the prior art enables activation of cellular factors, for instance RIG-1, in a photocontrolled manner and is simultaneously easily obtainable synthetically by known routine procedures for the automated synthesis of oligonucleotides including photocaged ribonucleoside and / or deoxyribonucleoside building blocks.

[0064] It has surprisingly been found that a conjugate according to the invention is easily obtainable synthetically and generally allows for the temporal and spatial controlled provision of the double-stranded oligonucleotide at a desired target location, where, upon activation (i.e. removal of the PPG(s) / photocage(s) by irradiation with light), it can perform its biological function as an activator of cellular factors, e.g., as a RIG-1 ligand inducing an immune response, as described herein. In other words, the present invention provides control over protein activity, such as RIG-1 activity, through doublestranded oligonucleotide-based molecules, e.g. dsRNA RIG-1 ligands, withphotocontrolled activity. Since the conjugate itself is basically comprised of naturally occurring biomolecules and inactive, e.g. unable to bind to and thereby activate RIG- I, simple systemic administration of the conjugate without or significantly reduced adverse side-effects is possible; unwanted activation of the immune system and any adverse effects associated therewith can be avoided or drastically decreased. This makes the conjugate of the present invention a versatile tool for many applications, including, for example, nucleic acid-based immunotherapy or an application as a vaccine adjuvant in a mRNA vaccine.

[0065] The conjugate of the present invention may also be part of a pharmaceutical composition or a vaccine, or may be for use as medicament, e.g., in the prevention or treatment of cancer or an infection (e.g. virus infection), or in a method of vaccination that results in defined and predictable induction of an innate immune response, preferably a consistent defined level of innate immune activation during vaccination whilst not negatively influencing vaccine-associated target antigen translation from mRNA.Conjugate

[0066] The present invention provides a conjugate as described herein and the attached claims. This conjugate comprises: (a) at least one double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and (b) at least one photoremovable protecting group (PPG); wherein

[0067] the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the at least one double-stranded oligonucleotide (a).Double-stranded oligonucleotide

[0068] The conjugate of the present invention comprises at least one double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand.

[0069] As used herein, the term “double-stranded" refers to the first and second strand of nucleotides, as defined herein, whose nitrogenous bases are joined by hydrogen bonding, typically via Watson-Crick base pairing rules. As a result, the double-stranded oligonucleotide comprised in the conjugate of the present invention comprises or consists of at least the indicated number of ribonucleotides, which form double-stranded RNA (dsRNA). This means that the double-stranded oligonucleotide may comprise further portions forming a double strand as described herein, e.g. doublestranded DNA (dsDNA) moieties. Said double-stranded oligonucleotide, e.g. dsRNA, may be self-complementary. However, any extension or portion not forming a double strand, such as, for example, an optionally present single-stranded nucleic acid overhang or non-nucleic acid based extension, is not considered to be part of the "double-stranded oligonucleotide".

[0070] The term "oligonucleotide" as used herein refers to a polynucleotide formed from a plurality of linked nucleoside units. The nucleoside units may be ribonucleosides and / or deoxyribonucleosides. Accordingly, an "oligonucleotide" can also include a "hybrid oligonucleotide" and / or "chimeric oligonucleotide".

[0071] 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).

[0072] 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).

[0073] In generally preferred embodiments the oligonucleotide may be a "RNA oligonucleotide". An "RNA oligonucleotide" is a type of "oligonucleotide" as described and discussed herein, which can include unmodified RNA as well as modified RNA (e.g. to improve efficacy or stability), and polymers of nucleoside surrogates. In a generally preferred embodiment the double-stranded oligonucleotide of the conjugate of the present invention is double-stranded RNA (dsRNA). Such dsRNA may have a biological activity, e.g. act as immunogen, like dsRNA that is most commonly found in certain viral genomes.

[0074] The inventors of the present invention have found out that the sequence of the double-stranded oligonucleotide, e.g. dsRNA, is not particularly limited. Said another way, the present invention does not require a specific sequence for the doublestranded oligonucleotide, e.g. dsRNA, but is essentially independent from the sequence of the double-stranded polynucleotide. The terms "oligonucleotide" and "polynucleotide" are synonyms and may be used interchangeably herein.

[0075] The term "unmodified RNA" refers to a molecule in which the components of the nucleic acid, namely sugar, base and phosphate moiety, are the same or essentiallythe same as that which occur in nature, preferably as occur naturally in the human body.

[0076] The term "modified RNA" refers to a molecule in which one or more of the component(s) of the nucleic acid, namely sugar, base, and / or phosphate moiety, is / are different from that which occur(s) in nature, preferably different from that which occur(s) in the human body; and also includes ribonucleic acid analogues, which are not ribonucleic acids, but structurally similar and able to perform the biological function of ribonucleic acids. While the latter may be referred to as modified "RNAs", they will of course, because of the modification, include molecules which are not RNAs.

[0077] The term "nucleoside surrogates" refers to 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.

[0078] All oligonucleotide or nucleic acid sequences listed herein are generally in the 5'- to 3'-direction unless otherwise indicated.

[0079] An oligonucleotide can be obtained from existing nucleic acid sources, including genomic or cDNA, but is 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 methylene phosphonate, 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).

[0080] The oligonucleotide 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. Oligonucleotides may be short, double-stranded RNA molecules, and include antisense oligonucleotides (ASO), RNA interference (RNAi), and aptamer RNAs.

[0081] In some embodiments of the conjugate of the present invention the doublestranded oligonucleotide may be a hybrid oligonucleotide. In some embodiments of the conjugate of the present invention the double-stranded oligonucleotide may be a chimeric oligonucleotide. In a generally preferred embodiment, the double-stranded oligonucleotide may be an RNA oligonucleotide, preferably comprising or consisting of a double-stranded ribonucleic acid (dsRNA) sequence.

[0082] It is also contemplated herein that the double-stranded oligonucleotide may comprise an overhang at at least one strand of said double-stranded oligonucleotide. Such an overhang may preferably comprise at least one guanosine.

[0083] In some embodiments of the conjugate of the present invention the first and second strand of the double-stranded oligonucleotide may form a blunt-end at the 5'-end of the first strand. This means that the first and second strand of the double-stranded oligonucleotide are equal in length at the 5'-end of the first strand, but that an overhang may optionally be present at least one strand of the opposite end, i.e. 3'-end of the first strand and / or 5'-end of the second strand, of said double-stranded oligonucleotide. The double-stranded oligonucleotide may comprise an entirely blunt-ended sequence, i.e. the first and second strand of the double-stranded oligonucleotide are equal in length and do not comprise an overhang. In a preferred embodiment of the present invention, the double-stranded oligonucleotide may be a blunt ended double-stranded RNA. In an alternative preferred embodiment of the present invention, the doublestranded oligonucleotide may be a double-stranded RNA with a length of 8 to 17 ribonucleotides comprising at least one triphosphate at the 5'-end of the first or second, and an overhang at at least the first or second strand, wherein the overhang comprises at least one guanosine.

[0084] The first or second strand of the double-stranded oligonucleotide comprised in the conjugate according to the invention may further contain self-complementary sequences and may form a hairpin, for example with the sequence 5'- GACCUAGCCUAAAACUAGGUC-3' (SEQ ID NO: 1). The self-complementary sequence may be a palindromic sequence. For example, 5'- AAAGAUCCGGAUCAAAA-3' (SEQ ID NO: 2).

[0085] In generally preferred embodiments, the double-stranded oligonucleotide of the conjugate of the present invention is capable of inducing an immune response. This means that the double-stranded oligonucleotide without photocages, or after traceless removal of the PPGs coupled to its first or second strand, has a length and sequence that can induce an immune response.

[0086] 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 cellactivation, 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 plasmacytoid dendritic cells (PDCs) and / or monocytes.

[0087] It is generally preferred for said double-stranded oligonucleotide that it is capable of inducing an anti-viral immune response.

[0088] 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, IL-6, CXCL- 10, and TNF.

[0089] It is further generally preferred for the double-stranded oligonucleotide that it is capable of inducing interferon (IFN)-production, preferably of inducing type l-IFN production.

[0090] “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 IFNa, IFNco, I FNs, IFNK and I FN|3 that activate the IFN-a receptor and may be produced, although not exclusively, by plasmacytoid dendritic cells, fibroblasts, leukocytes, and endothelial cells.

[0091] Advantageously, the double-stranded oligonucleotide may be capable of activating retinoic acid-inducible gene I (RIG-I). Further, the double-stranded oligonucleotide may be capable of binding the receptor of retinoic acid-inducible gene I (RIG-I).

[0092] “Retinoic acid-inducible gene I (RIG-I)” is an innate immune surveillance protein and member of the pattern recognition receptor family, which detects the intracellular presence of viral RNA 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.

[0093] 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. However, uncontrolled activation of RIG-I can have a negative impact on the translation of a protein antigen in mRNA vaccination or cause severe immunotoxic side effects such as allergic reactions or anaphylactic shock. Identifying RIG-I ligandswhose activity can be controlled, especially in a temporal and spatial manner, has therefore been a long felt but hitherto unmet need in the art, particularly in recent years, accelerated by the worldwide SARS-CoV2 pandemic, a coronavirus whose genome is recognized by RIG-I.

[0094] The inventors of the present invention surprisingly found that it is possible to control activation of cellular factors, in particular RIG-I activation, spatially and temporally with a conjugate according to the present invention as described and demonstrated herein, which provides photocontrolled RIG-I activation. This photocontrolled RIG-I activation is, for example, demonstrated through application of a conjugate to primary monocyte cultures using interferon IFN-a production as a readout of RIG-I activation efficacy, where irradiation was found to be essential for activation of RIG-I. In further examples, THP1-dual cells, which allow for indirect monitoring of IFN production via an interferon response factor (IRF)-linked luciferase reporter gene expression system, it is demonstrated that a conjugate according to the present invention can stimulate IFN expression only upon irradiation, i.e. in a completely photocontrolled manner, thus allowing for a temporal and spatial control of the RIG-I activation (see, e.g. Figures 6 and 7 and Examples). This finding was surprising, since it could neither be predicted nor expected from the prior art that photocontrolled activation of RIG-I is possible. As stated before, the prior art is entirely silent in this regard and does neither provide any enabling disclosure for the claimed modifications nor any suggestion of the properties of the claimed combination of features.

[0095] “RIG-I activation”, as used herein, refers to the binding of RIG-I to a suitable ligand, typically viral genomic RNA, the subsequent displacement of the inhibitory caspase activation and recruitment domains (CARDs) of RIG-I, the oligomerization of multiple RNA-bound RIG-I monomers, and the interaction between oligomerized RIG-I CARD domains and mitochondrial antiviral-signalling protein (MAVS), which induces gene expression, most importantly expression of type I IFNs, via kinase-based signalling pathways.

[0096] For the length of the double-stranded oligonucleotide comprised in the conjugate of the present invention, it is generally preferred that the double-stranded oligonucleotide has a length of from 8 to 50 ribonucleotides. In the case of the doublestranded oligonucleotide comprised in the conjugate of the present invention, the length of the oligonucleotide should be understood to be the length of the individual strands, e.g. the first strand of ribonucleotides and the second strand of a ribonucleotides, that form or can form complementary base pairs with each other. This means that any optional present overhang at at least one of the first or second strand is not taken into account when determining the length of the double-strandedoligonucleotide. It is more preferred for the double-stranded oligonucleotide that it has a length of from 9 to 40 ribonucleotides. It is even more preferred for the doublestranded oligonucleotide that it has a length of from 11 to 30 ribonucleotides. In some generally preferred embodiments the double-stranded oligonucleotide may have a length of 11 ribonucleotides. In alternative generally preferred embodiments the double-stranded oligonucleotide may have a length of 20 to 24 ribonucleotides.

[0097] Exemplary embodiments of a conjugate according to the present invention may, for example, comprise a dsRNA having sequences according to the following Table 1 below, wherein each sequence is given in 5'3'-direction:Table 1 :Photoremovable protecting group (PPG)

[0098] The conjugate according to the present invention comprises at least one photoremovable protecting group (PPG), which is coupled to a ribose at the 2'-O- position or a nucleobase of a nucleotide in the first or second strand of the at least one double-stranded oligonucleotide comprised in the conjugate.

[0099] The term "photoremovable protecting group" ("PPG") as used herein refers to a moiety or chemical group that can be removed via irradiation. Such chemical groups are known in the art and are also denoted photolabile, photocleavable, photoactivatable or photosensitive. Such a group provides a function in regard to the structure it is attached to, in particular a function to hide or hinder ("cage") a biological activity of the structure it is attached to. In regard to the conjugate of the present invention, the PPG hinders the functionality of the double-stranded oligonucleotide, such as, for example, its function as an activator of cellular factors, e.g., RIG-I, asdescribed herein, and the PPG may also be referred to as photocage or photocaging group.

[0100] The terms "PPG" and "photocage" are used interchangeably herein. Further, a conjugate comprising one or more PPG(s) may also be referred to as "photocaged" herein.

[0101] In general, any PPG coupled to a ribose at the 2'-O-position or a nucleobase, as described and defined herein, is tracelessly cleavable by irradiation, preferably irradiation with light, providing structures of molecules that comprise the pure doublestranded oligonucleotide, as described herein. Restoration of the pure double-stranded oligonucleotide, as described herein, by "uncaging" (i.e. traceless removal of PPG(s) by irradiation with light) enables the biological activity of the double-stranded oligonucleotide, such as, binding of the double-stranded oligonucleotide to and activation of RIG-I. Further, the cleavage results in the generation of the naturally present groups, i.e. a hydroxy (OH) group at the 2'-position and a nucleobase amino group, respectively, in the double-stranded oligonucleotide. The main PPG(s) scaffold(s) is / are a by-product of the cleavage reaction. The removal of the PPG(s) thus will release a biological active double-stranded oligonucleotide having, for example, uncompromised activity as RIG-I ligand. The PPG(s) consequently enable(s) a photocontrollable activation, i.e. spatial and temporal control over the doublestranded oligonucleotide's activity, of cellular factors, e.g., RIG-I, as described herein, similar to the natural process.

[0102] The at least one PPG (also referred to as "photocage" herein) can be coupled to various positions within the first strand and / or second strand of the double-stranded oligonucleotide comprised in the conjugate according to the invention. Said another way, the caging effect is not particularly limited to a specific position of the photocage in the double-stranded oligonucleotide sequence. Moreover, the caging effect also is not particularly limited to a specific number of photocages present in the first and / or second strand of the double-stranded oligonucleotide sequence. In this regard, the inventors of the present invention could show that coupling of a photocage to a moiety (i.e. 2'-O-position of a ribose or a nucleobase) in the first strand, to a moiety in the second strand, or to moieties in both the first and second strand provide caging effects. However, a more pronounced caging effect was generally observed when at least two PPGs were present.

[0103] The number of PPG(s) present in the conjugate is not particularly limited and the presence of several photocages in the first and / or second strand is tolerated and can be beneficial. For practical reasons (sterical limitations, additional difficulties and costs for synthesis, absence of additional benefits / effects, etc.), however, the number ofPPG(s) present may not be unlimited, and may, for example, be in a range of from 1 to 6, preferably 2 to 5, in the first and / or second strand of the double-stranded oligonucleotide sequence.

[0104] In exemplary embodiments, the conjugate according to the present invention may, for example, comprise two or more PPGs, each of which being independently coupled to a ribose at the 2'-O-position and / or nucleobase of a nucleotide in the first and / or second strand.

[0105] In the case of two or more PPGs present in the conjugate, it is generally preferred that only one PPG is present per position of the first or second strand in the doublestranded oligonucleotide sequence to which a PPG has been coupled to, i.e. a first photocage is either coupled to the ribose 2'-O-position or to the nucleobase of a first nucleotide in the first or second strand, a second PPG is either coupled to the ribose 2'-O-position or the nucleobase of a second nucleotide in the first or second strand, and so on. However, it is technically not excluded, that in certain embodiments an individual nucleotide constituent in the first or second strand may contain a first photocage at the ribose 2'-O-position and a second photocage at the nucleobase. As previously noted, photocages may be the same or different at each occurrence.

[0106] In exemplary embodiments of conjugates according to the present invention, where two or more PPGs are present, the conjugate may, for example, comprise at least one PPG coupled to a ribose at the 2'-O-position of a nucleotide in the first strand and at least one PPG coupled to a ribose at the 2'-O-position of a nucleotide in the second strand. In further exemplary embodiments of the conjugate, at least one PPG may be coupled to a ribose at the 2'-O-position of a nucleotide in the first or second strand and at least one PPG may be coupled to a nucleobase of a nucleotide in the first or second strand. As previously noted, while technically not excluded, it is generally preferred that an individual nucleotide constituent in the first or second strand does not contain more than one photocage. In another exemplary embodiment of the conjugate, each of the two or more PPGs present in the conjugate may be independently coupled to a ribose at the 2'-O-position of a nucleotide in the first strand, i.e. a first photocage is coupled to the ribose 2'-O-position of a first nucleotide in the first strand, a second PPG is coupled to the ribose 2'-O-position of a second nucleotide in the first strand, and so on. In a further exemplary embodiment of the conjugate, each of the two or more PPGs present in the conjugate may be independently coupled to a ribose at the 2'-O-position of a nucleotide in the second strand, i.e. a first photocage is coupled to the ribose 2'- O-position of a first nucleotide in the second strand, a second PPG is coupled to the ribose 2'-O-position of a second nucleotide in the second strand, and so on. In other embodiments of the conjugate, at least one PPG may be coupled to a nucleobase ofa nucleotide in the first strand and at least one PPG may be coupled to a nucleobase of a nucleotide in the second strand. It is also possible that in the conjugate, each of two or more PPGs may be independently coupled to a nucleobase in the first strand. In yet further embodiments of the conjugate, each of the two or more PPGs present in the conjugate may be independently coupled to a nucleobase in the second strand. In any event, the number and exact position of PPGs present in the conjugate is not particularly limited, and any number which results in a stable molecule is tolerated. In some preferred embodiments, the conjugates may comprise a specific sequence and / or one or more particular photocages at certain positions, as described herein.

[0107] In generally preferred embodiments of the conjugate, the PPG may at each occurrence be independently selected from the group of orf / 70-nitrobenzyl-based groups, orf / 7o-nitro-2-phenethyl-based groups, (coumarin-4-yl)methyl-based groups, atto390-derived-coumarine-groups, benzocoumarin-based groups, 2- thionated coumarin-based groups, 2-dicyanomethylene coumarin-based groups, COUPY-based groups, cyanine(Cy)-based groups, porphyrin-based metal containing groups, boron dipyrromethen (BODIPY)-based groups, xanthene-based groups, pyronin-based groups, arylmethyl and arylcarbonylmethyl-based groups, 1 ,4- benzoquinone-based groups, bimane-based groups and 10H-phenothiazine-based groups.

[0108] In further generally preferred embodiments of the conjugate, the PPG may at each occurrence be independently selected from the group comprising the following groups (a1) to (a36):(a1)lerein is Cl, OH, OCH3or NH2, d R1is H or CH3;(a2)lerein is H or Me;(a5)(a6)lerein is S or C(=O);lereinRi is H and R2 is Br; orR1 is CH3 and R2 is H;lereindenotes the point of attachment of R;lereinRi is H and R2is H;R1 is H and R2is CH3;R1 is CH3and R2is H; orR1 is CH3and R2is CH3;lereindenotes the point of attachment of R;lereinRi is CH, R2is CH3 and X' is TfR1 is N, R2is CH3 and X' is Tf orR1 is CH, R2is CeHi3 and X is Br;lereinRi is OCH3, R2 is H and R3 is O;R1 is OCH3, R2 is H and R3 is NH;R1 is OCH3, R2 is H and R3 is S;R1 is OCH3, R2 is H and R3 is NOH;R1 is OCH3, R2 is CN and R3 is O;R1 is OCH3, R2 is CN and R3 is S;R1 is NEt2, R2 is H and R3 is O;R1 is NEt2, R2 is H and R3 is S;R1 is NEt2, R2 is CN and R3 is O; orR1 is NEt2, R2 is H and R3 is C(CN)2;lereinRi is CH3 and R2 is Et;R1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3 and R2 is Cl; orR1 is CH3and R2is I;lereinR1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3and R2is CH3;orR1 is tBu and R2 is H;R = H or CH3wherein # denotes the point of attachment of the PPG to a photolabile self- immolative linker, a ribose 2'-O-position, a nucleobase N-atom or a nucleobase O-atom.

[0109] It is preferred that the one or more PPG(s) present in the conjugate according to the invention provide advantageous photoremovability upon irradiation, e.g. irradiation from an energy source providing electromagnetic radiation in a wavelength ranging of from 300 nm to 2500 nm, preferably irradiation with light in a range of from 350 nm to 800 nm, and more preferably with light in a range of from 365 nm to 625 nm. Depending on intended use, activation wavelength of the PPG (i.e the wavelength at which the photocage is removed from the double-stranded oligonucleotide by chemical reaction, typically a photolysis reaction) can be selected, and PPGs with different activation wavelengths' have been described and are generally known in the art. For example, PPGs that can be activated by irradiation with red light, i.e. electromagnetic radiation having a wavelength within a range of from 620 nm to 800 nm, preferably 620 nm to 750 nm, have, for example, been described by Wohlrabova, L. et al., 2024 and Mueller, P. et al. 2021. PPGs that can be activated by irradiation with green light, i.e. electromagnetic radiation having a wavelength within a range of from 495 nm to 620 nm, preferably 495 nm to 570 nm, have, for example, been described by Kaufmann, J. et al., 2022.Coupling of PPG

[0110] In the conjugate according to the present invention, at least one PPG is coupled to a 2'-O-position of a ribose or to a nucleobase of a nucleotide in the first or second strand of the double-stranded oligonucleotide.

[0111] Coupling of a PPG to the 2'-O-position of a ribose or a nucleobase (e.g. N-atom or O-atom) in a nucleoside as a precursor for routine automated synthesis of DNA and RNA has been widely described (see, e.g. Pitsch et al., 1999) and can be achieved by those skilled in the art of organic synthesis following routine methods and using commercially available standard materials and reagents of typical commercial grade. Examples for the synthesis of such precursors according to previously described methods are provided in Figure 2.

[0112] In general, coupling of a PPG to the 2'-O-position of a ribose or a nucleobase (e.g. N-atom or O-atom) of a nucleotide in the first or second strand of the double-stranded oligonucleotide comprised in the conjugate can be such that the PPG may be coupled to the 2'-O-position of a ribose or a nucleobase, either directly by a photocleavable bond or via a photolabile self-immolative linker.

[0113] The term "photocleavable bond" as used herein, is meant to refer generally to a direct attachment of the photocage either to the oxygen atom at the ribose 2'-O-positionor to a nitrogen atom or to an oxygen or nitrogen atom at a nucleobase by a covalent bond that is efficiently photochemically cleaved upon exposure to light resulting in the traceless removal of the PPG and restoration of the hydroxy group at the ribose 2'-O- position and of the amino moiety at a nucleobase, respectively.

[0114] The term "photolabile self-immolative linker" as used herein, is meant to refer generally to a covalent construct connecting the PPG with the ribose 2'-O-position or nucleobase atom, which is tailored to correlate the cleavage of two chemical bonds (i.e. the chemical bond between the linker and the PPG on one hand and the bond between the linker and the ribose 2'-0 or nucleobase atom it is attached to on the other hand) in the photocaged double-stranded oligonucleotide (inactive precursor) and designed to degrade spontaneously in response to activation of the PPG by irradiation with light. The linker is incorporated joining the trigger and the reporter moieties. The linker forms a scissile bond to the PPG group and a stable linkage to the ribose 2'-0 or nucleobase atom; however, upon activation of the PPG by light and subsequent cleavage, the coupling of the linker to the ribose 2'-0 or nucleobase atom is rendered labile, resulting in disassembly of the linker and release of the uncaged doublestranded oligonucleotide. The "photolabile self-immolative linker" is not particularly limited and a significant number of "photolabile self-immolative linkers" are known. Exemplary embodiments, where the PPG is coupled via a "photolabile self-immolative linker" can comprise a structure selected from the group of:wherein X represents the point of attachment of the linker to the 2'-O-position or the nucleobase, and RL1, RL2, RL3and RL4are as defined in any one of the following (ia) to (id):(ia) RL1is H, RL2is H, RL3is CH3and RL4is H;(ib) RL1is H, RL2is Br, RL3is CH3and RL4is Br;(ic) RL1is H, RL2is H, RL3is NO2 and RL4is H; or(id) RL1is CH3O, RL2is H, RL3is CH3O and RL4is CH3O.

[0115] In some preferred embodiments of the conjugate, the PPG(s) may be coupled to a ribose 2'-O-position via a photolabile self-immolative linker. In the case of coupling a PPG to a ribose 2'-O-position, it may be preferred that the PPG is coupled to the ribose 2'-O-position via a photolabile self-immolative linker. In other preferred embodiments of the conjugate according to the invention, the PPG(s) may be coupled to a nucleobase directly by a photocleavable bond or may be coupled to a nucleobase via a photolabile self-immolative linker. In case the PPG is coupled to a nucleobase, it is generally preferred that the PPG is coupled to a nitrogen atom (N atom) or oxygen atom (O atom) of the nucleobase.

[0116] In case when the conjugate according to the invention contains a nucleotide having a nucleobase with a coupled PPG, it is preferred that such a constituent comprises a structure selected from the group of:wherein * represents the coupled PPG and Rib represents the ribose moiety of the nucleotide.

[0117] In some embodiments, where the conjugate according to the invention contains a nucleotide having a nucleobase with a coupled PPG, said nucleotide may comprise the structure:

[0118] In case when the conjugate according to the invention contains a nucleotide having a PPG coupled to a ribose at the 2'-O-position, it is preferred that such a constituent comprises a structure selected from:whereinNB represents the nucleobase moiety of the nucleotide, such asDi- / Triphosphate

[0119] A diphosphate or diphosphate group (also referred to or depicted herein as “pp” or "PP") refers to a moiety of two phosphate groups (PO43) sequentially bound to the ribose sugar of a nucleotide molecule. A triphosphate or triphosphate group (also referred to or depicted herein as “ppp” or "PPP") refers to a moiety of three phosphate groups (PO43") sequentially bound to the ribose sugar of a nucleotide molecule.

[0120] In some generally preferred embodiments of the conjugate according to the present invention, the double-stranded oligonucleotide comprised in the conjugate comprises at least one diphosphate or triphosphate at the 5'-end of the first strand or the second strand, preferably a diphosphate or triphosphate at the 5'-end of the first strand, and more preferably a triphosphate at the 5'-end of the first strand.Modification(s)

[0121] The conjugate according to the present invention comprises at least one doublestranded oligonucleotide comprising: (i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and (ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with thefirst strand. In some embodiments of said conjugate, the first strand and / or the second strand of the double-stranded oligonucleotide may comprise at least one modification(s).

[0122] The term "modification" as used herein generally refers to at least one synthetic or artificial element in a molecule that constitutes a difference from the naturally occurring molecule. This may, for example, concern a base, sugar or phosphate moiety of a nucleotide in a nucleic acid molecule, such as, for example, replacement or elimination of one or more substituents at the base or sugar moiety, or substitution of the ribophosphate backbone.

[0123] Such at least one modification(s) comprised in the first and / or second strand of the at least one double-stranded oligonucleotide sequence(s) comprised in the conjugate of the invention may be selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O- methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester- modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate- morpholino-modification(s).

[0124] For the at least one modification(s), it has to be considered that in preferred embodiments of the double-stranded oligonucleotide sequence(s), the first and second strand are based on ribonucleic acid (RNA). 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.

[0125] The term "deoxy-modification(s)" as used herein generally refers to a modification such that the first and / or second strand of the at least one double-stranded oligonucleotide sequence comprises one or more (deoxy)ribonucleotide(s). Preferably, the 2'-hydroxy group is eliminated. Deoxygenation is a common nucleoside modification of RNA.

[0126] The term “O-methyl-group-modification(s)” as used herein means that the first and / or second strand of the at least one double-stranded oligonucleotide sequence 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.

[0127] The term “fluoro-group-modification(s)”, as used herein, means that the first and / or second strand of the at least one double-stranded oligonucleotide sequence is modified such that it comprises one or more fluoro-group(s). Preferably, it may be substituted to comprise one or two fluoro substituent(s) at the 2'-position. 2'-F modification of RNA, where the 2'-hydroxyl of the ribose moiety of a nucleoside is replaced by fluor, is common often resulting in increased binding affinity and nuclease resistance.

[0128] 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.

[0129] The term “phosphodiester-modification(s)" as used herein means that the first and / or second strand of the at least one double-stranded oligonucleotide sequence is modified such that it contains bonds where a non-bridging oxygen in the phosphodiester linkage of RNA has been replaced by sulfur resulting in phosphoro- thioate-bonds. Phosphoro-thioate-modification of mRNA is common and typically enhances translation efficiency thereby providing a strategy to improve mRNA functionality.

[0130] A peptide nucleic acid (PNAs) is a DNA / RNA analogue in which sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units, peptide nucleic acid modification(s) and phosphoro-diamidate-morpholino-modification(s). This structure provides for increased stability against enzymatic degradation. PNA nucleotides can be included in a DNA or RNA oligonucleotide thereby providing "peptide nucleic acid modification(s)"

[0131] The term "phosphoro-diamidate-morpholino-modification(s)" as used herein means that the first and / or second strand of the at least one double-stranded oligonucleotide sequence is modified such that it contains units where the sugarphosphate backbone is replaced by one or more phosphoro-diamidate-morpholino unit(s). Such phosphoro-diamidate-morpholino unit(s) are non-toxic, uncharged, not metabolised and can be included in a DNA or RNA oligonucleotide.Linear double-stranded oligonucleotide

[0132] In a preferred embodiment of the conjugate according to the present invention, the conjugate may comprise a double-stranded oligonucleotide having a length of from 8 to 50 ribonucleotides, preferably a length of from 9 to 40 ribonucleotides, more preferably of from 11 to 30 ribonucleotides, even more preferably of 11 ribonucleotides or 20 to 24 ribonucleotides. Such a double-stranded oligonucleotide may also be referred to as "linear double-stranded oligonucleotide" herein, i.e. the complementary base pairing of the bases of the first and second strand results in a double-stranded oligonucleotide with linear structure.

[0133] In an advantageous embodiment of a conjugate of the invention that comprises the linear double-stranded oligonucleotide, said linear double-stranded oligonucleotide may be blunt-ended. The term "blunt-ended" as used in connection with such a linear double-stranded oligonucleotide refers to a double-stranded nucleic acid sequence whose first and second strand are equal in length and does not contain an overhang as described or defined herein. In a preferred embodiment of the present invention, the linear double-stranded oligonucleotide comprised in the conjugate of the invention is double-stranded RNA (dsRNA), preferably blunt ended dsRNA capable of binding to and / or activating RIG-I.

[0134] In an advantageous embodiment of the conjugate of the invention, the linear double-stranded oligonucleotide comprised in the conjugate comprises at least one triphosphate at the 5'-end of at least the first or second strand, preferably at the 5'-end of the first strand. An exemplary embodiment of such a conjugate is shown in Figure 1.

[0135] It is further preferred that the first strand or the second strand of a linear doublestranded oligonucleotide as described in the foregoing paragraphs comprises at least one modification(s). The at least one modification(s) comprised in the first and / or second strand of said linear double-stranded oligonucleotide sequence may be selected from the group consisting of deoxy-modification(s), O-methyl-group- modification(s), preferably 2'-O-methyl-modification(s) or 2'-O-methoxyethyl- modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester- modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate- morpholino-modification(s).

[0136] Also in the case of a conjugate of the invention comprising the linear doublestranded oligonucleotide as described in the foregoing paragraphs, the conjugate comprises at least one PPG(s) that is / are coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of said linear double-stranded oligonucleotide. The at least one PPG(s) may be the same or different at eachoccurrence. In a generally preferred embodiment of the conjugate, said conjugate may comprise at least one PPG(s) coupled to the 2'-O-position by a photolabile self- immolative linker.

[0137] It may be preferred for the conjugate of the invention comprising the linear doublestranded oligonucleotide as described in the foregoing paragraphs and at least one PPG(s) coupled to the 2'-O-position of a nucleotide in the first or second strand of said linear double-stranded oligonucleotide by a photolabile self-immolative linker that this at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1 , position 2, position 4, position 5, position 6, position 7, position 8, position 9, position 14, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , position 12, position 13, position 24, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii). It may be further preferred that the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1 , position 5, position 6, position 7, position 8, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii).

[0138] In advantageous exemplary embodiments of the conjugate with the linear doublestranded oligonucleotide as described in the foregoing paragraphs, one PPG may be coupled at position 1 of the first strand, and at least one PPG(s) may, optionally, be coupled at:(i) a position of the first strand selected from position 5, position 6, position 7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof.

[0139] In alternative advantageous exemplary embodiments of the conjugate with the linear double-stranded oligonucleotide as described in the foregoing paragraphs, one PPG may be coupled at position 6 (in a 3' to 5' prime direction), one PPG may be coupled at position 7 (in a 3' to 5' prime direction) of the second strand of said linear double-stranded oligonucleotide, and at least one PPG(s) may, optionally, be coupled at:(i) a position of the first strand selected from position 1 , position 5, position 6, position 7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, and any combination thereof. In a preferred embodiment of such a conjugate, one PPG is coupled at position 6 (in a 3' to 5' prime direction) and one PPG is coupled at position 7 (in a 3' to 5' prime direction) of the second strand of said linear double-stranded oligonucleotide.

[0140] It may also be generally preferred for a conjugate of the invention comprising the linear double-stranded oligonucleotide as described in the foregoing paragraphs that it comprises at least one PPG(s) that may be coupled to a nucleobase of a nucleotide in the first or second strand of said linear double-stranded oligonucleotide by a photocleavable bond or via a photolabile self-immolative linker, preferably by a photocleavable bond. This means that the conjugate may comprise only PPG(s) that is / are coupled to a nucleobase or that it may comprise PPG(s) that is / are coupled to a nucleobase in addition to PPG(s) that is / are coupled to the 2'-O-position of a nucleotide in the first or second strand of said linear double-stranded oligonucleotide as described in the paragraphs above. As stated before, in case of two or more PPGs present in the conjugate, it is generally preferred that only one PPG is present per position of the first or second strand in the linear double-stranded oligonucleotide sequence to which a PPG has been coupled to.

[0141] In generally preferred embodiments of an inventive conjugate comprising at least one PPG(s) coupled to a nucleobase as described in the foregoing paragraph, the at least one PPG(s) may be coupled at:(i) a position of the first strand selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and / or (ii).

[0142] It may further be generally preferred for a conjugate of the invention comprising the linear double-stranded oligonucleotide and at least one PPG(s) that may be coupled to a nucleobase of a nucleotide in the first or second strand of said linear doublestranded oligonucleotide as described in the paragraphs above that said conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in the first and / or second strand. In the case of 2, 3, 4 or 5 coupled PPGs, it may further be preferred when, in each ofthe first or second strand of the linear double-stranded oligonucleotide sequence, the nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1 , 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase.

[0143] It may further be generally preferred for a conjugate of the invention comprising the linear double-stranded oligonucleotide and at least one PPG(s) that may be coupled to a nucleobase of a nucleotide in the first or second strand of said linear doublestranded oligonucleotide as described in the foregoing paragraphs that said conjugate comprises at least one PPG(s) that is coupled to a nucleobase at position 1 of the second strand and / or at position 2 of the second strand and / or at position 5 of the second strand, such as at positions 2 and 5 or 1 , 2 and 5 of the second strand, or at positions 3 and 5 of the second strand.Conjugate comprising oligonucleotide with overhang

[0144] As stated before, the conjugate of the present invention may optionally comprise that the double-stranded oligonucleotide comprised in the conjugate comprises an extension or overhang at at least one of the first or second strand of said doublestranded oligonucleotide. The terms “overhang” and "extension" are used interchangeably herein and generally refer to a non-double stranded sequence, as defined herein, in the conjugate, unless specifically defined otherwise.

[0145] In a generally preferred embodiment of the conjugate, the conjugate may comprise a linear double-stranded oligonucleotide as described above, wherein said linear double-stranded oligonucleotide portion has a length of from 8 to 17 ribonucleotides, at least one di- or triphosphate, preferably a triphosphate, at the 5'-end of at least the first or second strand, and further comprises a single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a di- / triphosphate, wherein the single-stranded overhang comprises at least one guanosine. In this embodiment of the conjugate of the present invention, an overhang may comprise a single-stranded nucleic acid sequence and / or a non-nucleic acid based portion, e.g. a fluorophore, sterol, vitamin or lipophilic substitution, at the above indicated termini of the first or second strand of said otherwise linear doublestranded oligonucleotide sequence. An exemplary embodiment of such a conjugate is shown in Figure 8A.

[0146] A suitable fluorophore in the context of the present invention may, for example, be FAM, FITC, cyanine dyes (Cy2, Cy3, Cy5) or any Atto or Alexa Fluor Dye.

[0147] A suitable lipophilic substitution in the context of the present invention may, for example, be a substitution with oleat or palmitat.

[0148] A suitable sterol in the context of the present invention may, for example, be cholesterol.

[0149] It is preferred for the conjugate comprising said guanosine containing singlestranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a di- / triphosphate that the linear double-stranded oligonucleotide portion has a length of from 8 to 15, 8 to 12, 10 to 12, or 11 ribonucleotides, preferably 10 to 12, or 11 ribonucleotides.

[0150] In a generally preferred embodiment of the conjugate comprising said guanosine containing single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a di- / triphosphate, the at least one di- / triphosphate may be at the 5'-end of the strand of said linear doublestranded oligonucleotide portion, which also comprises the single-stranded overhang.

[0151] In generally preferred embodiments, said single-stranded overhang comprising at least one guanosine has a length of at least 3 nucleotides. In this regard, it may be advantageous when said single-stranded overhang has a length of 3 to 15 nucleotides, 3 to 10 nucleotides, 3 to 8 nucleotides or 3 to 5 nucleotides. It may further be preferred when said single-stranded overhang comprises at least two guanosines or at least three guanosines. In one exemplary and preferred embodiment, the single-stranded overhang may have a length of 3 to 5 nucleotides and comprise at least three guanosines, such as three consecutive guanosines (GGG). For example, the singlestranded overhang may comprise or consist of 5 nucleotides having the sequence adenosine-adenosine-guanosine-guanosine-guanosine (AAGGG). The singlestranded 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.

[0152] In a generally preferred embodiment of the conjugate comprising said guanosine containing single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a triphosphate, the first and second strand of the linear double-stranded oligonucleotide portion may form at least a blunt-end at the 5'-end of the first strand. In this regard, it may be further advantageous when the 5'-end of the first strand has a triphosphate.

[0153] The conjugate comprising said guanosine containing single-stranded overhang as described above may also comprise a linear double-stranded oligonucleotide portion comprising a further overhang in addition to the guanosine containing single-stranded overhang. Such an additional overhang is preferably present at the strand of said linear double-stranded oligonucleotide which does not comprise the single-stranded overhang comprising at least one guanosine. Preferably, said further overhang maybe a single-stranded RNA-overhang or an overhang with a fluorophore, sterol, vitamin and / or lipophilic substitution.

[0154] In further generally preferred embodiments of the conjugate comprising said guanosine containing single-stranded overhang as described in the foregoing paragraphs, at least said linear double-stranded oligonucleotide portion may be connected with the guanosine containing single-stranded overhang via a linker moiety L.

[0155] In case when the conjugate comprises a further overhang in addition to the guanosine containing single-stranded overhang as described above, a linker moiety L may also connect said further overhang with the linear double-stranded oligonucleotide portion.

[0156] If both, the guanosine containing single-stranded overhang and the further overhang, are connected via a linker moiety L with the linear double-stranded oligonucleotide portion, the linker moiety L may be at each occasion independently selected. This means that the guanosine containing single-stranded overhang may be connected via a first linker moiety L and the further overhang may be connected via a second linker moiety L; the first and second linker moiety L may be the same or different from each other.

[0157] It may further be advantageous when the conjugate comprising said guanosine containing single-stranded overhang as described in the foregoing paragraphs comprises at least one modification(s). Preferably, the at least one modification(s) may be a modification as described herein. For example, the at least one modification(s) comprised in the conjugate, e.g. in the first and / or second strand of the linear doublestranded oligonucleotide sequence or the single-stranded overhang comprising at least one guanosine, may be selected from the group consisting of deoxy- modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl- modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro- diamidate-morpholino-m+odification(s).

[0158] An advantage of the conjugate comprising said guanosine containing singlestranded overhang as described in the foregoing paragraphs may be that this conjugate, due to its relatively short length, may be capable of penetrating cells without the presence of a transfection reagent. Accordingly, the conjugate comprising said guanosine containing single-stranded overhang may be characterized in that it is capable of penetrating cells without the presence of a transfection reagent.

[0159] 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.

[0160] Thus, the conjugate comprising said guanosine containing single-stranded overhang as described herein will be able to induce immune stimulation in tissues or cells that before were challenging to access.

[0161] For example, the conjugate comprising said guanosine containing single-stranded overhang may comprise a dsRNA of a sequence according to the following Table 2 below, wherein each sequence is given in3'-direction (those sequences according to SEQ ID NOs: 1 - are given in the respective sequence listing of the present application with “t” instead of “u” due to sequence listing program requirements):Table 2Linker L, BL1, BL2 and L3

[0162] The term “linker" and "linker moiety" are used interchangeably herein. As used in connection with linker moiety L, linker BL1 , linker BL2 and linker L3 herein, the term “linker" or "linker moiety" refers generally to a covalent construct connecting the indicated elements within the embodiments of the conjugate as described herein. This means that a "linker" or “linker moiety” has the primary function to connect at least two constituents of the conjugate as described herein without providing any inherent signalling or pharmacological function perse. It is generally preferred that a "linker" or "linker moiety" provides spatial freedom and enough flexibility so that a connected double-stranded oligonucleotide as provided herein can exert its biological activity as described herein, e.g. induce an immune response and / or RIG-l-activity.

[0163] Unless defined otherwise, the following remarks and explanations concerning linker moiety L (also referred to as linker L) are also readable upon and applicable to any of linker BL1 , linker BL2 and linker L3 described herein.

[0164] The linker moiety L may be a nucleotide-linker or a hydrocarbon-based linker.

[0165] In some preferred embodiments of the conjugate described herein, the linker L may be a nucleotide-linker. In case when the linker L is a nucleotide-linker, it may be preferred that the linker L has a length of at least 2 nucleotides. It may be further advantageous when the linker moiety L is a nucleotide-linker having a length of 2 to 10 nucleotides. In some preferred embodiments, the nucleotide-linker may be an adenosine-linker, preferably an adenosine-linker comprising at least 2 adenosines. In this regard, it may be more preferred when the adenosine-linker comprises or consists of 2 adenosines.

[0166] In some preferred embodiments of the conjugate described herein, the linker moiety L may be a hydrocarbon-based linker. Such a hydrocarbon-based linker may preferably comprise 3 to 24 main chain atoms, more preferably it may comprise 6 to 18 main chain atoms, more preferably it may comprise 8 to 16 main chain atoms, such as 10 to 14 main chain atoms or 12 main chain atoms.

[0167] The term “main chain atoms” as used herein refers to the atoms present in the backbone of the linker moiety, e.g. the linker moiety L. That backbone may be formed solely by carbon atoms (all main chain atoms are carbon atoms, i.e. only main chain carbon atoms are present), but in cases, where one or more O, N and / or S atoms are present or where carbon atoms have been replaced by O, N or S, the respective O, N or S atoms also count as main chain atoms for the total number of main chain atoms.

[0168] In case when the linker moiety L is a hydrocarbon-based linker, the linker moiety L may be a hydrocarbon-based linker according to Formula IFormula I wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7. For clarity and avoidance of doubt, in the hydrocarbon-based linker according to Formula I, both, each of the carbon-atoms of the (C2-C3)-alkylene as well as each O atom, count to the total number of main chain atoms.

[0169] In a generally preferred embodiment of a hydrocarbon-based linker moiety L, said linker-moiety L may be a polyalkylene-glycol-linker according to Formula IIFormula II wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7. In this embodiment, the total number of main chain atoms is n times (two carbon atoms plus one O atom). It may be advantageous when the linker moiety L is a polyalkylene- glycol-linker according to Formula II with n being 6.Conjugate comprising structure of Formula III, IV and V

[0170] In some generally preferred embodiments of the invention, the at least one doublestranded oligonucleotide comprised in the conjugate may comprise a structure according to Formula III, Formula IV or Formula V as depicted below:RNA1 - BL1 -RNA3 RNA1 RNA3RNA2 RNA4 RNA2 - BL2 -RNA4Formula III Formula IVRNA1 - BL1 - RNA3RNA2 - BL2 - RNA4Formula V whereinRNA1 represents the first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA2 represents the second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the first strand via complementary base pairing;RNA3 represents a third strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA4 represents a fourth strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the third strand via complementary base pairing;BL1 , if present, represents a bivalent linker that covalently bonds the 3'-end of RNA1 to the 3'- or 5'-end of RNA3;BL2, if present, represents a bivalent linker that covalently bonds the 5 -end of RNA2 to the respective 5'- or 3'-end of RNA4; whereinRNA1 and RNA2, as well as RNA3 and RNA4, each have no overhang of the 5’- terminal nucleotide residues, and wherein RNA1 and RNA2, as well as RNA3 and RNA4, each can comprise an overhang of the 3’-terminal nucleotide residues of not more than five nucleotides, preferably of not more than four or three nucleotides, more preferably of not more than two nucleotides, even more preferably of not more than one nucleotide, especially no overhang of the 3’-terminal nucleotide residues. Exemplary embodiments of conjugates comprising a structure according to Formula III, Formula IV or Formula V are shown in Figure 3.

[0171] For clarity and avoidance of doubt, it is noted that in the structures according to Formula III, Formula IV or Formula V, the at least one double-stranded oligonucleotide comprised in the conjugate of the invention can be the dimer unit formed by RNA1 (a first strand of a ribonucleic acid having a length of at least eight nucleotides) and RNA2 (a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand) or the dimer unit formed by RNA3 and RNA4. Accordingly, when the conjugate of the invention comprises a double-stranded oligonucleotide having a structure according to Formula III, Formula IV or Formula V, the at least one (PPG) that is comprised in said conjugate will, for example, be coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in RNA1 (the first strand) or RNA2 (the second strand) as described herein before.

[0172] In the conjugate comprising at least one double-stranded oligonucleotide having a structure according to Formula III, Formula IV or Formula V as described above, the linker BL1 and BL2 can independent from each other selected from the group of a nucleotide-linker as described above, a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms as described above, a phosphodiester linker (OPO(OH)O-), and any combination thereof. In case when BL1 is absent, the 3 -end of RNA1 may be directly bonded to the 3'- or 5'-end of RNA3. In case when BL2 is absent, the 5 -end of RNA2 may be directly bonded to the respective 5'- or 3'-end of RNA4.

[0173] In generally preferred embodiments of structures according to Formula III, Formula IV or Formula V, the sequence of RNA1 may be identical to the sequence of RNA3.

[0174] In generally preferred embodiments of structures according to Formula III, Formula IV or Formula V, the sequence of RNA2 may be identical to the sequence of RNA4.

[0175] It is further generally preferred that in some embodiments of structures according to Formula III, Formula IV or Formula V, each of RNA1 , RNA2, RNA3 and RNA4 may have a length of from 9 to 40 nucleotides, preferably of from 11 to 30 nucleotides, more preferably of from 20 to 24 nucleotides.

[0176] In alternative generally preferred embodiments of structures according to Formula III, Formula IV or Formula V, each of RNA1 , RNA2, RNA3 and RNA4 may have a length of from 8 to 11 nucleotides, preferably 11 nucleotides.

[0177] In case when the conjugate comprises a structure of Formula III, the structure of Formula III may advantageously be configured such that BL1 may bond the 3'-end of RNA1 to the 3'-end of RNA3, and that RNA1 and RNA3 each may have, at their 5'- end, a di- / triphosphate, preferably a triphosphate.

[0178] In case when the conjugate comprises a structure of Formula IV, the structure of Formula IV may advantageously be configured such that BL2 may bond the 5'-end of RNA2 to the 5'-end of RNA4, and that RNA1 and RNA3 each may have, at their 5'- end, a di- / triphosphate, preferably a triphosphate.

[0179] In case when the conjugate comprises a structure of Formula V, the structure of Formula V may advantageously be configured such that BL1 may bond the 3'-end of RNA1 to the 3'-end of RNA3, BL2 may bond the 5'-end of RNA2 to the 5'-end of RNA4, and that RNA1 and RNA3 each may have, at their 5'-end, a di- / triphosphate, preferably a triphosphate.

[0180] In case when the conjugate comprises a structure of Formula V, the structure of Formula V may also advantageously be configured such that BL1 may bond the 3'-end of RNA1 to the 5'-end of RNA3 and BL2 may bond the 5'-end of RNA2 to the 3'-end of RNA4, wherein the strands of the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 are not complementary, and wherein RNA1 and RNA4 each may have, at their 5'-end, a di- / triphosphate, preferably a triphophosphate.

[0181] In generally preferred embodiments of structures according to Formula III, Formula IV or Formula V as described herein, the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 may have the same or a different length. In this regard, it is noted that a dimer unit RNA1 / RNA2 as well as a dimer unit RNA3 / RNA4 represents a dsRNA as described hereinbefore. This means that a dimer unit RNA1 / RNA2 or a dimer unit RNA3 / RNA4 may, within the limits indicated above, have a structure comprising or consisting of a dsRNA as described herein above, for example, a dsRNA embodimentof a linear double-stranded oligonucleotide or a dsRNA embodiment of the doublestranded oligonucleotide with a single-stranded overhang, which overhang comprises at least one guanosine. Further, RNA1 , RNA2, RNA3 and RNA4 each represent a continuous coherent strand. Thus, RNA1 , RNA2, RNA3 and RNA4 each have a 3' terminus and a 5' terminus and there is no interruption of the sequence in RNA1 , RNA2, RNA3 and RNA4. However, RNA1 , RNA2, RNA3 and RNA4 each may comprise one or more modification(s), such as, for example, RNA analog nucleotides or RNA derivative nucleotides as described in US 2018 / 195063 A1.

[0182] In a generally preferred embodiment of the conjugate according to the invention, the double-stranded oligonucleotide having a structure according to Formula III, Formula IV or Formula V is capable of inducing an immune response and / or to activate RIG-I.

[0183] It is further generally preferred that in embodiments of structures according to Formula III, Formula IV or Formula V as described herein, the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 each may be blunt-ended double-stranded RNA. This means, as noted before, RNA1 and RNA2, as well as RNA3 and RNA4, each may form at least one blunt-end at the 5'-end of RNA1 or RNA3; or, more preferably, RNA1 and RNA2, as well as RNA3 and RNA4, each have no overhang of the 5’- and 3'- terminal nucleotide residues.

[0184] In generally preferred embodiments of structures according to Formula III, Formula IV or Formula V as described herein, at least one of RNA1 , RNA2, RNA3 and RNA4 may comprise at least one modification(s). The at least one modification(s) of at least one of RNA1 , RNA2, RNA3 and RNA4 may be selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl- modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro- diamidate-morpholino-modification(s).PPG(s) in Formula III, Formula IV or Formula V

[0185] As stated above, the at least one (PPG) comprised in the above described conjugate according to the invention, which comprises a structure according to Formula III, Formula IV or Formula V as double-stranded oligonucleotide, is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in RNA1 or RNA2. In accordance with the invention one or more further PPG(s) may be present in such a conjugate comprising a structure according to Formula III, Formula IV or Formula V as double-stranded oligonucleotide. Said one or more further PPG(s) may be present ineach of RNA1 and RNA2, as well as RNA3 and RNA4. For example, one or more further PPG(s) may be present in RNA1 and / or RNA2, as, for example, generally described in section "Photoremovable protecting group (PPG)" above.

[0186] In a generally preferred embodiment of the conjugate comprising a structure according to Formula III, Formula IV or Formula V as described in the foregoing paragraphs, the conjugate may be such that, in addition to RNA1 and / or RNA2, at least one of RNA3 and RNA4 comprises at least one PPG(s).

[0187] It is further generally preferred for the conjugate comprising a structure according to Formula III, Formula IV or Formula V as described herein that the at least one PPG(s) in RNA1 , RNA2, RNA3 and / or RNA4 may be the same or different at each occurrence.

[0188] In a preferred embodiment of the conjugate comprising a structure according to Formula III, Formula IV or Formula V as described herein, the at least one PPG(s) comprised in at least one of RNA1 , RNA2, RNA3 and / or RNA4 may be coupled to 2'- O-position via a photolabile self-immolative linker as described herein before. In such a conjugate, the at least one PPG(s) may be preferably coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1 , position 5, position 6, position 7, position 8, position 9, and any combination thereof;(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , and any combination thereof; or(iii) two or more PPGs are coupled to RNA1 , RNA2, RNA3 and RNA4 at positions selected from (i) and (ii).It may be further preferred in such a conjugate that one PPG may be coupled at position1 of RNA1 and / or RNA3, and at least one PPG(s) may, optionally, be coupled at:(i) a position of RNA1 and / or RNA3 selected from position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , and any combination thereof.In an alternative preferred embodiment of such a conjugate, one PPG is coupled at position 6 (in a 3' to 5' prime direction), one PPG is coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA4, and at least one PPG(s) may, optionally, be coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1 , position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, position 11 and any combination thereof. For example, one PPG may be coupled at position 6 (in a 3' to 5' prime direction) and one PPG may be coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA4.

[0189] In a preferred embodiment of the conjugate comprising a structure according to Formula V, wherein BL2 may bond the 5 -end of RNA2 to the 3'-end of RNA4 and the strands of the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 are not complementary, as described herein, at least one PPG(s) may be coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1 , position 5, position 6, position 7, position 8, position 9, and any combination thereof;(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , and any combination thereof; or(iii) two or more PPGs are coupled to RNA3 and / or RNA4 at positions selected from (i) and (ii).For example, in such a conjugate, one PPG may be coupled at position 1 of RNA1 and / or RNA4, and at least one PPG(s) may, optionally, be coupled at:(i) a position of RNA1 and / or RNA4 selected from position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , and any combination thereof.In another preferred example of such a conjugate, one PPG may be coupled at position 6 (in a 3' to 5' prime direction), one PPG may be coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA3, and at least one PPG(s) may, optionally, be coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1, position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, position 11 , and any combination thereof. For example, one PPG may be coupled at position 6 (in a 3' to 5' prime direction) and one PPG may be coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA3.

[0190] In a further preferred embodiment of the conjugate comprising a structure according to Formula III, Formula IV or Formula V as described herein above, at least one PPG(s) may be coupled to a nucleobase by a photocleavable bond or via a photolabile self-immolative linker, preferably by a photocleavable bond. Stated differently, a conjugate comprising a structure according to Formula III, Formula IV orFormula V as described herein above may be characterized in that the at least one PPG(s) comprised in at least one of RNA1 , RNA2, RNA3 and / or RNA4 may be coupled to a nucleobase by a photocleavable bond or via a photolabile self-immolative linker, preferably by a photocleavable bond. This means that such a conjugate may solely comprise one or more PPG(s) coupled to a nucleobase in any one of RNA1 , RNA2, RNA3 and / or RNA4. However, the conjugate can also comprise one or more PPG(s) coupled to a 2'-O-position of a nucleotide in at least one of RNA1 , RNA2, RNA3 and / or RNA4 as described herein before, in addition to PPG(s) coupled to one or more nucleobases of nucleotide(s) in RNA1 , RNA2, RNA3 and / or RNA4. In case when the conjugate comprises both types of coupled PPG(s), it is generally preferred that only one PPG is present per position in RNA1 , RNA2, RNA3 and RNA4 to which a PPG has been coupled to.

[0191] In case when the conjugate comprising a structure according to Formula III, Formula IV or Formula V as described herein above is a conjugate that comprises at least one PPG(s) coupled to a nucleobase, it is preferred when the at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof.

[0192] It is generally preferred for a conjugate of the invention which comprises a structure according to Formula III, Formula IV or Formula V and at least one PPG(s) coupled to a nucleobase as described herein above that the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in one or more of RNA1 , RNA2, RNA3 and RNA4, and wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1 , 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase.

[0193] It is further generally preferred for a conjugate of the invention which comprises a structure according to Formula III, Formula IV or Formula V and at least one PPG(s) coupled to a nucleobase as described herein above that the conjugate is configured such that at least one PPG(s) is / are coupled at position 1 and / or at position 2 and / or at position 5, such as at positions 2 and 5 or positions 1 , 2 and 5, of RNA2 and / or RNA4, or at positions 3 and 5 of RNA2 and / or RNA4.

[0194] For a conjugate comprising at least one PPG(s) coupled to a nucleobase and a structure according to Formula V, wherein BL2 may bond the 5 -end of RNA2 to the 3'-end of RNA4 and the strands of the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 are not complementary, as described herein above, it may be preferred that the at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof.For said embodiment of the conjugate, it may further be preferred that the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in one or more of RNA1 , RNA2, RNA3 and RNA4, and wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1 , 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase. It is generally advantageous when at least one PPG(s) is / are coupled at position 1 and / or at position 2 and / or at position 5, such as at positions 2 and 5 or positions 1, 2 and 5, of RNA2 and / or RNA3, or at positions 3 and 5 of RNA2 and / or RNA3.Conjugate comprising structure of Formula VI

[0195] In a generally preferred embodiment of the invention, the conjugate may comprise a structure according to Formula VI as depicted below:Formula VI wherein sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of a mRNA strand;L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms; anddsO represents a double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and (ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand. Exemplary embodiments of conjugates comprising a structure according to Formula VI are shown in Figure 4.

[0196] For clarity and avoidance of doubt, it is again noted that in the structure according to Formula VI, the at least one double-stranded oligonucleotide comprised in the conjugate of the invention is the unit shown as dsO. Accordingly, when the conjugate of the invention comprises a double-stranded oligonucleotide having a structure according to Formula VI, the at least one PPG(s) that is comprised in said conjugate will be coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the dsO as described herein before.

[0197] In a generally preferred embodiment of a structure according to Formula VI, the single-stranded nucleic acid sequence (sN) may be complementary to at least one nucleic acid sequence comprised in the mRNA strand. It may further be preferred for the sN that it is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand, such as a 3'- or 5'- untranslated region (UTR)-nucleic acid sequence or poly-A-tail in the mRNA strand. Particularly preferable, the sN is capable of hybridizing to said 3'- or 5'-UTR-nucleic acid sequence or poly-A-tail of the mRNA-strand via Watson-Crick-base-pairing.

[0198] In a generally preferred embodiment of a structure according to Formula VI, the dsO may be a linear double-stranded oligonucleotide or a double-stranded oligonucleotide with a guanosine containing single-stranded overhang as described herein above. It may further be particularly preferred for the dsO that it is a dsRNA, preferably a dsRNA capable of binding to and / or activating RIG-I. Such a dsRNA may be blunt-ended or comprise an overhang, such as a RNA-overhang or an overhang with a fluorophore, lipophilic substitution, sterol or vitamin as described herein before. Preferably, the dsO may further comprise at least one triphosphate. In case when the dsO comprises or consists of a dsRNA, that dsRNA may comprise at least one triphosphate at at least one strand of said dsRNA, preferably at least one triphosphate at the 5'-end of the first or second strand of said dsRNA, more preferably at the 5'-end of the first strand of the dsRNA.

[0199] It is preferred for a conjugate of the invention which comprises a structure according to Formula VI that linker L3 directly connects the sN with the dsO. As stated previously, the term “linker" refers generally to a covalent construct connecting the sN and dsO constituents of the structure according to Formula VI without providing any inherent signalling or pharmacological function per se. It is generally preferred thatlinker L3 provides spatial freedom and enough flexibility so that the dsO as described herein can exert its biological activity, e.g. induce an immune response and / or RIG-I- activity. The linker L3 may be a nucleotide-linker or a hydrocarbon-based linker as described above. In case when linker L 3 is a nucleotide-linker, it may be preferred that the linker L3 has a length of at least 2 nucleotides, such as a length of 2 to 10 nucleotides. For example, the linker L3 may be an adenosine-linker, such as an adenosine-linker comprising or consisting of 2 adenosines. In case when linker L3 is a hydrocarbon-based linker, it may preferably comprise 3 to 24 main chain atoms, preferably 6 to 18 main chain atoms, more preferably 8 to 16 main chain atoms, such as 10 to 14 main chain atoms or 12 main chain atoms as described herein. In a preferred embodiment of the hydrocarbon-based linker, linker L3 may have a structure according to Formula IFormula I wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7 as described herein before. It may further be preferred that linker L3 has a structure according to Formula IIFormula II wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7, more preferably n may be 6. In a generally preferred embodiment, L3 may connect the 3'-end of the first strand or the 5'-end of the second strand with the sN.

[0200] In a generally preferred embodiment of the conjugate of the invention which comprises a structure according to Formula VI, the conjugate enables temporally and spatially controlled, targeted activation of RIG-1, for example, as an immune adjuvant, such as a vaccine adjuvant in the context of mRNA vaccination as described herein. Accordingly, a conjugate comprising a structure according to Formula VI may be an immune adjuvant or for use as an immune adjuvant.mRNA vaccine

[0201] A mRNA vaccine typically consists of mRNA molecules that encode a desired antigenic protein that is capable of stimulating an immunogenic reaction, initiating a process that ends with the generation of acquired immunity and therefore protection against future infection of the relevant pathogen(s). This is achieved through the local cytoplasmic translation of vaccine mRNA to produce the antigenic protein(s), which is typically then incorporated into the cell membrane and / or secreted.

[0202] In mRNA vaccines, the mRNA may be constructed so as to guarantee optimal translation of the antigen. This requires the insertion of a cap structure (N7-methyl- guanosine 5’-5’-linked to the first nucleotide of the mRNA) at the 5’-end.

[0203] For example, when administered intramuscularly, mRNA vaccines typically lead to adaptive immune system activation via initial transfection of muscle cells, epidermal cells, and tissue-resident antigen presenting cells, e.g. dendritic cells and macrophages, thereby initiating priming of T and B cells. Later, vaccine particles will typically move from the injection site to secondary lymphoid tissue via lymphatic drainage. Transfection of lymphoid tissue antigen presenting cells initiates priming and activation of both T and B cells, contributing to generation of adaptive immunity. This adaptive immunity is mediated by, amongst other cell types, memory B cells, plasma cells, and T helper cells.

[0204] In addition to adaptive immune responses, innate immune activation is key to successful immunization following administration of an mRNA vaccine. Non-self RNA is a potent activator of the innate immune system, with host RNA undergoing extensive intracellular modification to prevent innate immune recognition by clearly differentiating self and non-self RNA. This property means that RNA can serve as both an indirect immunogen by way of protein expression, as described herein, and an adjuvant in the composition of vaccines.

[0205] 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.

[0206] Adjuvant activity requires activation of the innate immune system, acting to enhance vaccine immunogenicity and degree of protection conferred by vaccination. Currently approved mRNA vaccines are believed to activate the innate immune system by virtue of the presence of contaminant RNA particles that are a by-product of themethod of production. Although these contaminants are able to activate the innate immune system and thus act as adjuvants, the lack of control over their quantity, quality, and method of action means that mRNA vaccines will benefit from the invention of defined adjuvants that may be co-administered. Such adjuvants may be adjusted in strength and / or mechanism of immune activation to best meet the needs of a given mRNA vaccine construct. The conjugate as described herein, upon activation, may be able to serve as an adjuvant. In this regard, a conjugate comprising a linear doublestranded oligonucleotide or an oligonucleotide comprising an overhang, or a conjugate comprising a structure according to Formula III, IV, V or VI, capable of activating RIG- I, an innate immune receptor involved in innate immune recognition of RNA viruses, upon activation, may be preferred and can function as adjuvant, such as a vaccine adjuvant.

[0207] A vaccine adjuvant may mean any material, which is incorporated with a vaccine for the purpose of enhancing the immune response of the vaccine. This is typically an innate immune response. This can be a contaminant from the manufacturing process, such as bacterial lipopolysaccharide, inorganic compounds such as aluminium salts, or defined immune stimulants, such as cytokines, e.g. interleukin-1. In many cases, adjuvants function by mimicking molecules that are termed pathogen-associated molecular patterns (PAMPs), evolutionarily conserved molecular motifs that act to indicate that a cell or molecule is non-self and must therefore evoke an immune response.

[0208] Initial activation of the innate immune system is a necessary step in mRNA-vaccine elicited adaptive immunity. Hence, mRNA vaccines are typically dependent upon vaccine adjuvants for their functionality. RNA can elicit innate immune activation via various endosomal and cytosolic immune receptors. For example, non-host RNA binds and activates, amongst others, toll-like receptor 3 (TLR3) and 7 (TLR7), melanoma differentiation-associated protein 5 (MDA5), and retinoic acid-inducible gene I (RIG-I). Activation of these receptors drives type I interferon (IFN I) responses and plays a key role in innate antiviral responses.

[0209] Due to the potential adverse effects that systemic innate immune activation could drive, many mRNA vaccines have been modified to attempt to evade innate immune recognition of the antigen-encoding mRNA. For example, replacement of uridine with naturally occurring uridine-derivatives, such as pseudo-uridine, which is part of the processing that allows host mRNA to avoid stimulating innate immune receptors, or addition of a 2’-O-methyl group onto the first nucleotide to prevent binding of RIG-I.

[0210] Despite steps taken to avoid cytoplasmic RNA sensing when preparing mRNA for use in vaccine compositions, it is likely that mRNA vaccines stimulate the innateimmune system, in part, via unintended nucleic acid contaminants generated during the manufacturing process, e.g. RNA by-products. This draws a parallel to the presence of minute quantities of microbial contaminants that act as adjuvants in protein-antigen-based vaccines, a now well-characterized and necessary element of vaccine.

[0211] The exact nature of the adjuvant-contaminants that allow for successful immunization via mRNA vaccines remains unclear, as few studies have studied the activation of the innate immune system in vivo following vaccination with approved mRNA vaccines. However, it is clear that a robust antiviral and IFN-I response is elicited just one day post-vaccination, providing a clear indication that the mRNA vaccine triggers direct innate immune activation.

[0212] Despite the efficacy of said mRNA vaccines, it is readily apparent that reliance on undefined, non-controlled contaminants to elicit a key step in vaccine-induced immunity is undesirable. Future mRNA-based vaccines would therefore benefit from two elements - improved purification and / or synthesis to limit undefined contaminants from RNA manufacturing processes and, more importantly, the provision of well- defined adjuvants that can not only be adapted in both strength and nature, but precisely controlled in space and time, to best elicit the required immune reaction without causing undesired immunotoxic side effects such as allergic reactions or anaphylactic shock.As mentioned before, a conjugate according to the present invention comprising a structure according to Formula VI enables temporally and spatially controlled, targeted activation of RIG-I, which elicits an immune response, and can therefore also function as mRNA vaccine adjuvant. In addition, one or more said conjugate(s) may be part of a mRNA vaccine, such as, for example, shown in Figure 5.Conjugate comprising structure of Formula VII

[0213] In a generally preferred embodiment of the invention, the conjugate may comprise a structure according to Formula VII as depicted below:Formula VII wherein mRNA is a mRNA-strand;(sN-L3-dsO) represents a unit comprising (a) to (c), wherein(a) sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand;(b) L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms;(c) dsO represents a double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and(ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand; and m is an integer in the range of from 1 to 4. As mentioned previously, a schematic representation of an exemplary embodiment of an uncaged conjugate having a structure according to Formula VII is shown in Figure 5, depicting the structure of both the mRNA- strand encoding an immunogen and four units (sN-L3-dsO) capable of activating RIG-I that are hybridized to the mRNA-strand.

[0214] For clarity and avoidance of doubt, it is again noted that in the structure according to Formula VII, the at least one double-stranded oligonucleotide comprised in the conjugate of the invention is the element indicated as dsO in the depicted unit (sN-L3- dsO). Accordingly, when the conjugate of the invention comprises a double-stranded oligonucleotide having a structure according to Formula VII, the at least one PPG(s) that is comprised in said conjugate will be coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the dsO as described herein before.

[0215] It is preferred that in an embodiment of the conjugate according to the invention comprising a structure according to Formula VII that the conjugate is a nucleic acid-construct. It is more preferred that this embodiment of the conjugate of the present invention can be an engineered nucleic acid-construct. The term “engineered” as used herein may mean that such a construct was artificially made and does not occur as such in nature. It is further generally preferred that this embodiment of a conjugate according to the present invention comprises at least two units (sN-L3-dsO), i.e. m = 2; more preferably said conjugate comprises two, three or four units (sN-L3-dsO), and most preferably said conjugate comprises four units (sN-L3-dsO). It is further generally preferred, as mentioned previously, that each unit (sN-L3-dsO) is able to function as RIG-l-ligand as described herein.

[0216] It is further generally preferred for the conjugate of the invention comprising a structure according to Formula VII that in each unit (sN-L3-dsO) each of the individual elements sN, L3 and dsO can be independently selected. It is also generally preferred that each of the individual elements sN, L3 and dsO, respectively, can, in each unit (sN-L3-dsO), be the same or different at each occurrence. For instance, a conjugate may comprise at least two units (sN-L3-dsO) with identical elements sN, L3 and dsO or two units (sN-L3-dsO) which differ in at least one of elements sN, L3 or dsO, such as, for example, two units having a different sN, but identical elements L and dsO. In some embodiments, a unit (sN-L3-dsO) may comprise or consist of a structure according to Formula VI. mRNA

[0217] Messenger RNA (mRNA) is a single-stranded RNA molecule that results from transcription of DNA by RNA polymerase. The initial mRNA transcript is then processed to produce a mature mRNA molecule that may be read by the ribosome to produce a polypeptide (protein). A mRNA may be part of a conjugate according to the invention, such as in the conjugate having a structure according to Formula VII, or can be any other mRNA that may be chosen as target for hybridization for the sN.

[0218] It is preferred that the mRNA comprises a coding sequence of a protein, a peptide or an antigen, preferably an antigen that is able to induce an immune response or a cancer antigen. Thus, the mRNA is able to encode one or more relevant antigen(s) via its coding sequence.

[0219] A "coding sequence" as used herein generally refers to a sequence of mRNA that encodes a protein or polypeptide.

[0220] The term “protein” is equally used herein with the term "polypeptide", and also includes protein fragments, preferably biologically active protein fragments, in particular protein fragments that are able to induce an immune response, as well as peptides. A "protein fragment" as used herein generally refers to a peptide or proteinthat results from cleavage or destruction of a larger protein. A "peptide" as used herein is a protein molecule which consists of two or more, and typically 30 or less, amino acids linked to each other via one or more covalent peptide bonds of the type -OC-NH- and having a chain and / or cyclic ring structure of amino acids. The term "polypeptide" as used herein describes a molecule, which, for example, comprises or consists of more than 30 amino acids with two or more of them being coupled to each other via a covalent peptide bond. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or nonidentical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo or heterotrimers etc. The terms "polypeptide" and "protein" may also refer to naturally modified polypeptides / proteins, wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

[0221] As used herein and in the context of the present invention, the term “antigen” means a substance that can stimulate the immune system to produce a set of specific antibodies and that combines with the antibody through a specific binding site or epitope.

[0222] A cancer antigen as used herein and in the context of the present invention may mean an antigen produced by a tumor cell. Cancer antigens are therefore useable as biomarkers and may be treatment targets for immunotherapy-based cancer treatment.

[0223] Expression of the mRNA may result in expression of said antigen(s) in transfected cells, with subsequent cell surface externalization and / or secretion resulting in recognition of said antigen by appropriate immune cells.

[0224] In some embodiments, the mRNA may be non-replicating and may simply encode the required antigen(s). In other embodiments, the mRNA may also encode viral replication machinery that results in intracellular amplification of said mRNA, thereby amplifying antigen expression.

[0225] In the present invention, the mRNA itself may typically have no intrinsic immune- stimulatory activity in the absence of uncaged, hybridized or conjugated complementary units (sN-L3-dsO) that are preferably capable of functioning as RIG-I ligand(s). The inventors of the present invention demonstrate this in Figure 10, which shows that an mRNA, when applied in the absence of photo-activated, hybridized RIG- I ligands, induces little to no expression of markers of immune activation.

[0226] It is generally preferred that the mRNA-strand, which may be used synonymously to “(the) mRNA” herein, may contain one or more untranslated regions (UTR). This isa region of the mRNA transcript that is not translated into protein. Such a region may be before the coding region (5’-UTR) or after it (3’-UTR). Both, the 5’-UTR and the 3’- UTR, may serve as the site of hybridization between the mRNA-strand and the sN element of a unit (sN-L3-dsO) or structure according to Formula VI. It is therefore preferred that the sN is complementary to at least one nucleic acid sequence comprised in mRNA / the mRNA strand, and particularly preferred that the sN is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand, preferably the 5'-UTR or 3'-UTR region of the mRNA-strand.

[0227] It is further preferred that the non-coding nucleic acid sequence of the mRNA- strand is or comprises a 3'-untranslated region (UTR)-nucleic acid sequence or a 5'- untranslated region (UTR)-nucleic acid sequence or a poly-A-tail.

[0228] As used herein, the term "poly-A tail" refers to a region of mRNA at the 3’-end of an mRNA sequence that exclusively contains adenine ribonucleotides. The region is non-coding, but controls mRNA processing and export.

[0229] As used herein, the term “non-coding nucleic acid sequence” refers to a region of DNA or RNA in the mRNA that is not translated during protein translation.

[0230] Non-coding regions of mRNA are often found between the 5’-end of the mRNA strand and the start codon of the coding sequence, termed the 5’-untranslated region (5’-UTR), or between the stop codon of the coding sequence and the 3’-end of the mRNA, termed the 3’-untranslated region (3’-UTR).

[0231] It is preferred that the mRNA-strand comprises such a 3'-untranslated region (UTR)-nucleic acid sequence and that the sN, as comprised in unit (sN-L3-dsO) or a structure according to Formula VI, is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 3'-untranslated region (UTR)-nucleic acid sequence. Further, it is preferred that the mRNA-strand comprises a 5'- untranslated region (UTR)-nucleic acid sequence and that the sN is capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand comprised in said 5'-untranslated region (UTR)-nucleic acid sequence. More preferably, the sN, as comprised in unit (sN-L3-dsO) or a structure according to Formula VI, may be capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'- untranslated region (UTR)-nucleic acid sequence of the mRNA-strand via Watson- Crick-base-pairing.

[0232] It may be advantageous for the target mRNA / mRNA-strand to comprise at least one modification(s). Such a modification(s) may preferably be selected from: pseudouridine-modification(s), preferably Nl-methyl-pseudouridine-modification(s), methoxyuridine-modification(s), preferably 5-methoxyuridine-modification(s),methylcytosine-modification(s), preferably 5-methylcytosine-modification(s), and methyl-adenosine-modification(s), preferably N6-methyl-adenosine-modification(s) and any combinations thereof. This means that the target mRNA / mRNA-strand may comprise one or more modification(s). These modification(s) may be identical or different at each modified position. Further, each of the one or more modification(s) that may be comprised in the target mRNA / mRNA-strand can be independently selected. According to the invention, it may, for example, be preferred for the target mRNA / mRNA-strand that it comprises at least one pseudo-uridine-modification(s). sN

[0233] As mentioned before, sN, a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of mRNA, is an element of a conjugate having the structure according to Formula VI as well as of the unit (sN-L3- dsO) comprised in a conjugate having the structure according to Formula VII. The mRNA may be any target mRNA. The term "target mRNA" as used herein generally refers to any mRNA to which the sN can hybridize to. The sN mediates hybridization between the dsO, e.g. dsRNA capable of binding to and / or activating RIG-1, and the target mRNA. Preferably, the target mRNA is the mRNA-strand as comprised in a conjugate having the structure according to Formula VII.

[0234] According to the invention it is further generally preferred that said single-stranded nucleic acid sequence sN can be complementary to at least one nucleic acid sequence comprised in the target mRNA, such as the mRNA-strand comprised in the conjugate having the structure according to Formula VII.

[0235] As stated before, a target mRNA, such as the mRNA-strand of Formula VII, may preferably comprise one or more non-coding regions, such as UTRs, e.g. 3'- or 5'- UTRs, or a poly-A-tail. In generally preferred embodiments of the sN, the sN is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the target mRNA. It may be further preferred that the sN is capable of hybridizing to a non-coding nucleic acid sequence of the target mRNA, which is or comprises a 3'-UTR nucleic acid sequence, a 5'-UTR nucleic acid sequence, or a poly- A-tail as described above.

[0236] It is generally preferred that hybridization of the sN to the target mRNA occurs via Watson-Crick base pairing, i.e. the formation of hydrogen bonds between nucleotide bases such that adenine pairs with uracil / thymine and guanine with cytosine. In certain preferred embodiments, the sN may hybridize to a 3'-UTR nucleic acid sequence or a 5'-UTR nucleic acid sequence in the target mRNA via Watson-Crick base pairing.

[0237] It is further generally preferred for the sN that it may have a sequence length in the range of from 5 to 15 nucleotides (also referred to herein as "nt"), more preferably a length in the range of from 8 to 13 nt.

[0238] In case when L3 is absent, i.e. when the dsO and the sN are directly connected, the sN may be attached to the 3'-end of the first strand (of the dsO) or to the 5'-end of the second strand of the dsO, preferably the sN may be attached to the 3'-end of the first strand, more preferably to the 3'-end of the first strand having a triphosphate at its 5'-end.L3

[0239] As noted before, L3 is a linker that can be an element of a conjugate having the structure according to Formula VI as well as of the unit (sN-L3-dsO) comprised in a conjugate having the structure according to Formula VII. If present, said linker L3 connects the herein described sN with the herein described dsO. As such, L3 may be a bivalent linker. Preferably, L3 has the general functionality of a linker as described above.

[0240] According to the invention it is generally preferred that L3 provides spatial freedom and enough flexibility so that the dsO as described herein can exert its biological activity, e.g. induce an immune response and / or RIG-l-activity.

[0241] L3 may be a nucleotide-linker or a hydrocarbon-based linker as described above.

[0242] L3 as defined herein comprises 3 to 24 main chain atoms. The term "main chain atoms" as used herein and noted before generally refers to the atoms of the linker that are present or form the backbone of the linker, and consist of atoms from the group consisting of: carbon (C), nitrogen (N), oxygen (O) and sulfur (S). It is further generally preferred that L3 may comprise 6 to 18 main chain atoms.

[0243] In case when L3 is a nucleotide-linker, such as an adenosine-linker, it may be preferred that L3 has a length of at least 2 nucleotides, such as a length of 2 to 10 nucleotides. It may be generally preferred that L3 is an adenosine-linker comprising or consisting of 2 adenosines.

[0244] In case when L3 is a hydrocarbon-based linker, it may have a structure according to Formula IFormula Iwherein n is an integer in the range of from 3 to 8, preferably in the range of from5 to 7 as described herein before; or a structure according to Formula IIFormula II wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7, more preferably n may be 6; as described herein before. As noted above, the total number of main chain atoms in such linkers is 3 to 24, preferably 6 to 18, more preferably 8 to 16 main chain atoms, such as 10 to 14 main chain atoms or 12 main chain atoms.

[0245] In a generally preferred embodiment, L3 may connect the 3'-end of the first strand of the dsO or the 5'-end of the second strand of the dsO, preferably the 3'-end of the first strand of the dsO, with the sN. dsO

[0246] According to the invention, the dsO as comprised in a conjugate having a structure according to Formula VI or in a conjugate having a structure according to Formula VII, comprises: (i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and (ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand. Within the indicated length limits, the dsO is not particularly limited and can be any doublestranded oligonucleotide however structured. In a generally preferred embodiment, the dsO may comprise a double-stranded nucleic acid sequence that, after uncaging (i.e. upon irradiation resulting in the traceless removal of the one or more PPG(s)), is capable of inducing an immune response, and / or to bind to and / or to activate RIG-I, preferably to bind to and activate RIG-I. For clarity and avoidance of doubt, it is further noted again that in the dsO, the at least one PPG(s) is coupled to a ribose at the 2'-O- position or a nucleobase of a nucleotide in the first or second strand of the dsO.

[0247] In further generally preferred embodiments, the dsO may be a linear doublestranded oligonucleotide as described herein above or a hereinbefore described double-stranded oligonucleotide comprising a single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand whichdoes not have a triphosphate, wherein the single-stranded overhang comprises at least one guanosine. In this regard, it may be particularly preferred that the dsO comprises or consists of a dsRNA, preferably a dsRNA that, after uncaging, may be capable of inducing an immune response, and / or to bind to and / or to activate RIG-1.

[0248] In some embodiments, the dsO may further comprise an overhang at the first or second strand as described herein before. In this regard, it may be preferred that the overhang is a RNA overhang or an overhang with a fluorophore, sterol, vitamin or lipophilic substitution.

[0249] In generally preferred embodiments, the dsO may have a length in the range of from 9 to 40 nucleotides, preferably of from 11 to 30 nucleotides, more preferably of from 20 to 24 nucleotides.

[0250] In alternative generally preferred embodiments, the dsO may have a length in the range of from 8 to 11 nucleotides, preferably 11 nucleotides.

[0251] According to the invention, it may be further generally preferred that the first and / or second strand of the dsO comprise(s) at least one modification(s). In this regard it may be preferred that the first strand of the dsO comprises at least one modification(s) at its 3'-end. It may further be generally preferred that the second strand of the dsO comprises at least one modification(s) at its 5'-end. It may be generally preferred for the dsO that both, the first and second strand, comprise at least one modification(s). It is generally preferred that the at least one modification(s) of the first and / or second strand of the dsO may be selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O- methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester- modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate- morpholino-modification(s) and any combination thereof. According to the invention, it may, for example, be advantageous that the second strand of the dsO comprises at least one phosphorothioate-modification(s). It may further be advantageous when the first strand comprises at its 3'-end and / or the second strand at its 5'-end at least one modification(s), preferably at least one O-methyl-group-modification(s), more preferably at least one 2'-O-methyl-group-modification(s), and even more preferably such modification(s) at the first and second strand of the dsO.Pharmaceutical composition

[0252] The present disclosure further provides a pharmaceutical composition comprising the conjugate of the present invention as defined in the claims and described herein.That composition may further comprise one or more component(s), such as a pharmaceutically acceptable carrier, an excipient or a delivery agent.

[0253] In some embodiments, the delivery agent is a complexation agent which forms a complex with the conjugate, double-stranded oligonucleotide or a precursor thereof, and facilitates the delivery of the conjugate into cells.

[0254] The complexation agent may be a polymer, preferably a cationic polymer, e.g. a cationic lipid; polyethylenimine (PEI); a collagen derivative, a biodegradable microsphere such as a liposome or poly (D,L-lactide-co-glycolide) copolymer (PLGA) microsphere; SNALPs; or ISCOMATRIX<> (CSL Limited).

[0255] 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.

[0256] SNALPs stand 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.

[0257] ISCOMATRIXo is made from saponin, cholesterol and phospholipids under defined conditions and forms cage-like structures typically 40 nm in diameter. ISCOMATRIXo has the dual capability of facilitating cargo (e.g., antigen) delivery and stimulating the immune system, both the cellular and humoral immune response.

[0258] The pharmaceutical composition of the present invention may further comprise another agent such as an agent that stabilizes the double-stranded oligonucleotide, in particular, dsRNA oligonucleotide, e.g., a protein that complexes with the oligonucleotide agent to form an RNP. 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.

[0259] The carrier may be, for example, a solid, liquid, or gas. Suitable carriers preferably are liquid and correspond to the substances ordinarily employed in formulation technology for pharmaceutical formulations.

[0260] The pharmaceutical composition may be produced under sterile conditions using standard pharmaceutical techniques well known to those skilled in the art.

[0261] 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, theconjugate may be in the form of a liquid composition, such as a solution, which may include convenient pharmaceutical media, such as, for example, water, buffers, glycols, oils, alcohols and the like. This form may be preferred for administration via inhalation.

[0262] 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, the oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.

[0263] In some embodiments, it is preferred that the pharmaceutical composition does not comprise any transfection reagent(s) or complex(es) or composition(s) thereof. The transfection reagent mentioned for those embodiments means such a transfection reagent as defined herein above. In some embodiments, administration routes may include administration via intranasal spray.

[0264] The pharmaceutical composition, upon administration and activation of the conjugate, may generate a large amount of type I IFN, in particular, IFN-a, in vitro and / or in vivo. The type I IFN, in particular, IFN-a, can be generated at high quantities from different cellular sources, including both immune and non-immune cells, from different species of vertebrates.Drug delivery system

[0265] The present disclosure further provides a drug delivery system comprising a conjugate as described herein or defined in the claims.

[0266] Said drug delivery system may comprise the conjugate according to the invention as described above, characterized in that the conjugate is a caged drug comprising the double-stranded oligonucleotide as therapeutic agent that is rendered biologically inactive by being coupled to at least one PPG(s); wherein said double-stranded oligonucleotide is capable of being activated and / or released in response to a predetermined wavelength and / or intensity of light which removes the at least one coupled PPG(s) tracelessly.

[0267] The drug delivery system may further comprise a marker. The marker may be any marker that is typically used in drug delivery systems, preferably the marker may be luminescent, such as, for example, a fluorophore as described herein.

[0268] The drug delivery system can be for medical uses as described herein below, such as for use in a method of treatment or prevention of a disease as described herein. For example, the drug delivery system may be for use in a method of treatment or prevention of a disease, comprising: administering the system to a patient; localisingthe system at the target site, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site.

[0269] The drug delivery system may be administered orally or parenterally to the patient. Parental administration may include intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing.In vitro application

[0270] The present application provides the in vitro use of the conjugates described above. In particular, the present application provides the use of at least one conjugate of the present disclosure for inducing an anti-viral response, in particular, a type I IFN response, more specifically, an IFN-a / p or CXCL-10 response; an immune response; or activity as RIG-I ligand, in particular, binding to and activating RIG-I, after uncaging, in vitro. The present application also provides the use of at least one conjugate as described herein or defined in the claims for inducing apoptosis of a tumor cell, after uncaging, in vitro.

[0271] The present disclosure further provides an in vitro method for stimulating RIG-I activity, an immune response or an anti-viral response, in particular, a type I IFN response, more specifically, an IFN-a, IFN-p or I P10 response in a cell, comprising the steps of: (a) contacting a cell with at least one conjugate as defined in the claims and as described above, wherein the cell expresses RIG-I, and (b) irradiating the cells.

[0272] The cells may express RIG-I endogenously and / or exogenously from an exogenous nucleic acid (RNA or DNA). The exogenous DNA may be a plasmid DNA, a viral vector, or a portion thereof. The exogenous DNA may be integrated into the genome of the cell or may exist extra-chromosomally. The cells include, but are not limited to, primary immune cells, primary non-immune cells, and cell lines. Immune cells include, but are not limited to, peripheral blood mononuclear cells (PBMC), plasmacytoid dendritric cells (PDC), myeloid dendritic cells (MDC), macrophages, monocytes, B cells, natural killer cells, granulocytes, CD4+T cells, CD8+T cells, and NKT cells. Non-immune cells include, but are not limited to, fibroblasts, endothelial cells, epithelial cells such as keratinocytes, and tumor cells. Cell lines may be derived from immune cells or non-immune cells.

[0273] The present disclosure also provides an in vitro method for inducing apoptosis of a tumor cell, comprising the steps of: (a) contacting a tumor cell with at least one conjugate as defined in the claims and as described above, and (b) irradiating the cells. The tumor cell may be a primary tumor cell freshly isolated from a vertebrate animal having a tumor or a tumor cell line. Alternatively, the cell may also be a virus infected cell.Medical uses

[0274] As mentioned before, a conjugate according to the present invention may be for use as a medicament.

[0275] The use as a medicament may further comprise directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide at the target site, wherein the activated or released double-stranded oligonucleotide is comprising directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged doublestranded oligonucleotide at the target site, wherein the activated or released doublestranded oligonucleotide is capable of inducing apoptosis of a tumor cell, of inducing an anti-viral immune response, of inducing binding to and / or activation of cytosolic helicase retinoic acid-inducible gene I (RIG-1), and / or of inducing IFN production, preferably of inducing type I IFN production.

[0276] A conjugate according to the present invention may also be for use in a method of treatment or prevention of a disease.

[0277] The method of treatment or prevention of a disease may comprise: administering the conjugate to a patient; localising the conjugate at the target site, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site.

[0278] The conjugate may be administered orally or parenterally to the patient. Parental administration may include intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing.

[0279] The disease that may be treated or prevented may be cancer or an infection, preferably a virus infection. Thus, the conjugate of the present disclosure may be for use in a method of treatment or prevention of a virus or viral infection.

[0280] 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 more conditions 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.

[0281] 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.

[0282] 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), HIV-AIDS, poliovirus, encephalomyocarditis virus (EMCV) and small-pox 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).

[0283] In case the disease is cancer, the cancer may preferably be selected from the group consisting of medulloblastoma, retinoblastoma, Hodgkin's lymphoma, oralcancer, 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.

[0284] In some embodiments, the disease may be a hematological tumor or a solid tumor.Conjugate for use in a method of infiltrating immune cells

[0285] Also provided herein is the conjugate as defined in the claims and described herein for use in a method of infiltrating immune cells, preferably T cells.

[0286] The infiltration, upon activation of the conjugate, may result in a stimulation of the immune cells. Accordingly, the conjugate may also be for use in a method of stimulating immune cells. Thus, the conjugate, upon activation as described herein, may have immunostimulatory activity.

[0287] As used herein, the term "immunostimulatory activity" refers to the capability of an agent, such as a molecule or a composition, e.g. the activated double-stranded oligonucleotide comprised in the conjugate, pharmaceutical composition or drug delivery system described herein, to induce an immune response. Thus, the present application provides a conjugate, pharmaceutical composition or drug delivery system capable of immunostimulatory activity, in particular anti-viral responses, in particular type l-IFN-production.

[0288] 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, e.g. the activated double-stranded oligonucleotide comprised in the conjugate, pharmaceutical composition or drug delivery system described herein, to induce IFN- a production from a cell capable of producing IFN-a.

[0289] 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.Conjugate for use in a method of turning cold into hot tumours

[0290] Also provided herein is the conjugate as defined in the claims and described herein 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). As noted previously, the conjugate enables a photocontrollable activation, i.e. spatial and temporal control over the doublestranded oligonucleotide's activity, of cellular factors, e.g., RIG-1, as described herein, and thus also enables immunological activation of surrounding cells of the tumor microenvironment by irradiation, which converts cold tumors into hot ones and allows the body's own immune system to help fight the cancer. Thus, the present application provides a conjugate that makes therapy-resistant, non-immunologically active tumors ("cold" tumors") accessible to treatment.Vaccine

[0291] The present application further provides a conjugate comprising a structure according to Formula VII as defined in the claims and described herein as mRNA vaccine and / or for use as a mRNA vaccine. Such mRNA vaccine may comprise or consist of one or more conjugate(s) comprising the structure according to Formula VII. As explained above, a mRNA vaccine typically consists of mRNA molecules that encode a desired antigenic protein that is capable of stimulating an immunogenic reaction, initiating a process that ends with the generation of acquired immunity and therefore protection against future infection of the relevant pathogen(s). This is achieved through the local cytoplasmic translation of vaccine mRNA to produce the antigenic protein(s), which is typically then incorporated into the cell membrane and / or secreted. Preferably, the conjugate having a structure according to Formula VII comprises such a vaccine mRNA in the mRNA-strand.Adjuvant

[0292] The present application further provides a conjugate as adjuvant and / or for use as an adjuvant, preferably as vaccine adjuvant, more preferably as mRNA vaccine adjuvant. As mentioned before, an adjuvant activates or helps to activate the innate immune system. The conjugates described herein, upon activation by irradiation, are able to activate RIG-I, an innate immune receptor involved in innate immune recognition of RNA viruses. RIG-I inter alia induces a response of the innate immune system, i.e. activates or helps to activate the innate immune system. In other words, the conjugate allows to control activation of the innate immune system via RIG-I inspace and time. Accordingly, the present application provides a conjugate capable of functioning as adjuvant. Thus, a conjugate as adjuvant and / or for use as adjuvant is provided. In this regard, it may be generally preferred that the conjugate may be a conjugate comprising a linear double-stranded oligonucleotide or an oligonucleotide comprising an overhang, or a conjugate comprising a structure according to Formula III, IV, V or VI, capable of activating RIG-1, an innate immune receptor involved in innate immune recognition of RNA viruses, upon activation.

[0293] In a further preferred embodiment, the conjugate comprising or consisting of a structure according to Formula VI as defined in the claims and described herein may be an adjuvant and / or for use as an adjuvant, preferably as vaccine adjuvant, more preferably as mRNA vaccine adjuvant.

[0294] Adjuvants are often used to modify or augment 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.

[0295] In a preferred embodiment, the conjugate may be a vaccine adjuvant or immune adjuvant. The term “immune adjuvant” as used herein means a substance that increases or modulates the immune response, and in particular the immune response to a vaccine.

[0296] It is further preferred that the conjugate may be a mRNA vaccine adjuvant as described above. In this regard, it is noted again that the conjugate can be advantageously adapted to hybridize to a sequence of a vaccine mRNA, and further enables to elicit the required immune reaction without causing undesired immunotoxic side effects such as allergic reactions or anaphylactic shock via temporally and spatially controlled, targeted activation of RIG-1. Moreover, strength and nature of the immune response can be controlled by the amount of conjugate applied or number of hybridized units (sN-L3-dsO). Thus, the conjugate is able to serve as adjuvant, for example, as a vaccine adjuvant or immune adjuvant, preferably as a mRNA vaccine adjuvant.Use of conjugate in the manufacture of a medicament

[0297] Provided herein is also the use of a conjugate as defined in the claims and described herein in the manufacture of a medicament for the treatment or prevention of a disease.

[0298] The disease may be cancer or an infection, preferably a virus infection. In case that 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.

[0299] The disease may also be a hematological tumor or a solid tumour.Conjugate for use in a method of inducing an immune response

[0300] As mentioned before, the conjugate, upon activation by irradiation with light, can induce an immune response, e.g. via activation of RIG-1. Thus, the present application also provides the conjugate as defined in the claims and described herein for use in a method of inducing an immune response.

[0301] The conjugate may be an antigen recognizing conjugate or part of an antigen recognizing conjugate or may be comprised in an antigen recognizing conjugate. In this regard, the antigen recognizing conjugate 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.

[0302] 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.

[0303] 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 iscomprised of a heterodimer having a and p chains (also known as TCRa and TCRp, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively).

[0304] Chimeric antigen receptors (CARs) - also known as chimeric immuno-receptors, chimeric T cell receptors or artificial T cell receptors - are receptor proteins that have been engineered 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.Method of treatment

[0305] The present application moreover provides a method of treating a disease, comprising the step of administering a therapeutically effective amount of the conjugate, drug delivery system or pharmaceutical composition, as defined in the claims and / or described herein, to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site.

[0306] In generally preferred embodiments, the disease may be cancer or an infection, preferably a virus infection.

[0307] 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 tumor (PNET)), and combinations thereof.

[0308] In alternative preferred embodiments, the disease may be a hematological tumor or a solid tumor.

[0309] In accordance with the method, the conjugate may be administered in a multimerized form. This means that the conjugate may form a multimer such that at least two, e.g. three or four of such conjugates may be administered as a complex.

[0310] The therapeutically effective amount of the conjugate may be administered to the subject in need thereof by any means known in the art, including, but not limited to,oral or parenteral routes. In case when the therapeutically effective amount of the conjugate is administered to the subject in need thereof parenterally, this may include intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing. It may be generally preferred that the therapeutically effective amount of the conjugate may be administered to the subject in need thereof via subcutaneous, intramuscular, intravenous, intra-parenteral, oral or intranasal route(s).

[0311] For oral administration, the conjugate may be provided in the form of a tablet or capsule, as a powder or granules, or as an aqueous solution or suspension.

[0312] 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.

[0313] 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.

[0314] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the conjugate 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. In accordance with the invention, aqueous suspensions may include a suspending agent such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0315] The therapeutically effective amount of the conjugate may also be administered as encapsulated formulations to protect the conjugate 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 willbe 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.

[0316] A suitable dose or therapeutically effective amount of the conjugate 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).

[0317] The step of administering a therapeutically effective amount of the conjugate may be once per day, or the conjugate may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the conjugate 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.

[0318] 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.

[0319] Toxicity and therapeutic efficacy of the conjugate, drug delivery system and pharmaceutical composition comprising the conjugate, as defined in the claims and described herein, 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 thepopulation). 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.

[0320] 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 conjugate, drug delivery system and pharmaceutical composition comprising the conjugate, as defined in the claims and described, herein 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 form employed and the route of administration utilized. For any conjugate used in the method of treatment provided herein, 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 conjugate 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.

[0321] The administering physician can adjust the amount and timing of the administration of the conjugate, drug delivery system and pharmaceutical composition comprising the conjugate, as defined in the claims and described herein, on the basis of results observed using standard measures of efficacy known in the art or described herein.Method of inducing an immune response

[0322] The present application further provides a method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the conjugate, drug delivery system or pharmaceutical composition comprising the conjugate, as defined in the claims and described herein, to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide as immune stimulant at the target site.

[0323] In generally preferred embodiments, the immune response may be induced by stimulating retinoic acid-inducible gene I (RIG-I).

[0324] The conjugate may have or comprise an immune adjuvant function.

[0325] In accordance with the method, the conjugate may be a conjugate having a structure according to Formula VII and may comprise:(a) at least one unit (sN-L3-dsO) capable of functioning as RIG-l-ligand;(b) at least two units (sN-L3-dsO) capable of functioning as RIG-l-ligands;(c) at least three unit (sN-L3-dsO) capable of functioning as RIG-l-ligands; or(d) at least four unit (sN-L3-dsO) capable of functioning as RIG-l-ligands.

[0326] In some embodiments, the immune response may be induced by activating retinoic acid-inducible gene I (RIG-1). In some embodiments, the conjugate may be administered in a multimerized form as described above.

[0327] In accordance with the method of inducing an immune response, it is preferred that the therapeutically effective amount of the conjugate, drug delivery system or pharmaceutical composition is administered by a route as described above in connection with the method of treatment. That is, administration may be by any means known in the art, including, but not limited to, oral or parenteral routes. The definitions given above with regard to administration routes also apply to the method of inducing an immune response. Consequently, the embodiments for the method of treatment as described above are readable upon and applicable to the method of inducing an immune response.Kit

[0328] The present application also provides a kit for use in medicine comprising the conjugate as defined in the claims and described herein.

[0329] 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.

[0330] 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, 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. The disease may also be a hematological tumor or a solid tumour.Items

[0331] The invention will be further illustrated by the following, non-limiting items:A conjugate comprising:(a) at least one double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and(ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary base pairs with the first strand; and(b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand. The conjugate according to item 1 , wherein the conjugate comprises two or more PPGs, each of which being independently coupled to a ribose at the 2'-O-position and / or nucleobase of a nucleotide in the first and / or second strand. The conjugate according to item 2, wherein at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the first strand and at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the second strand. The conjugate according to item 2, wherein at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the first or second strand and at least one PPG is coupled to a nucleobase of a nucleotide in the first or second strand. The conjugate according to item 2, wherein each of the two or more PPGs is independently coupled to a ribose at the 2'-O-position of a nucleotide in the first strand. The conjugate according to item 2, wherein each of the two or more PPGs is independently coupled to a ribose at the 2'-O-position of a nucleotide in the second strand. The conjugate according to item 2, wherein at least one PPG is coupled to a nucleobase of a nucleotide in the first strand and at least one PPG is coupled to a nucleobase of a nucleotide in the second strand. The conjugate according to item 2, wherein each of the two or more PPGs is independently coupled to a nucleobase in the first strand.The conjugate according to item 2, wherein each of the two or more PPGs is independently coupled to a nucleobase in the second strand. The conjugate according to any one of the preceding items, wherein the at least one double-stranded oligonucleotide comprises at least one diphosphate or triphosphate at the 5'-end of the first strand or the second strand; preferably at least one diphosphate or triphosphate at the 5'-end of the first strand; more preferably at least one triphosphate at the 5'-end of the first strand. The conjugate according to any one of the preceding items, wherein the first and second strand of the at least one double-stranded oligonucleotide form a blunt- end at the 5'-end of the first strand. The conjugate according to any one of the preceding items, wherein the first strand and / or the second strand of the at least one double-stranded oligonucleotide comprises at least one modification(s). The conjugate according to item 12, wherein the at least one modification(s) of the first strand and / or the second strand of the at least one double-stranded oligonucleotide is / are selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O- methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro- group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester- modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate- morpholino-modification(s). The conjugate according to any one of the preceding items, wherein the PPG(s) may be the same or different at each occurrence. The conjugate according to any one of the preceding items, wherein the PPG(s) is / are coupled to the 2'-O-position of a ribose or a nucleobase, either directly by a photocleavable bond or via a photolabile self-immolative linker. The conjugate according to item 15, wherein the PPG(s) is / are coupled to a ribose 2'-O-position via a photolabile self-immolative linker.17. The conjugate according to item 15, wherein the PPG(s) is / are coupled to a nucleobase, either directly by a photocleavable bond or via a photolabile self- immolative linker.18. The conjugate according to any one of the preceding items, wherein the PPG is at each occurrence independently selected from the group of ortho-nitrobenzyl- based groups, ortho-nitro-2-phenethyl-based groups, (coumarin-4-yl)methyl- based groups, atto390-derived-coumarine-groups, benzocoumarin-based groups, 2-thionated coumarin-based groups, 2-dicyanomethylene coumarin-based groups, COUPY-based groups, cyanine(Cy)-based groups, porphyrin-based metal containing groups, boron dipyrromethen (BODIPY)-based groups, xanthene-based groups, pyronin-based groups, arylmethyl and arylcarbonylmethyl-based groups, 1 ,4-benzoquinone-based groups, bimanebased groups and 10H-phenothiazine-based groups.19. The conjugate according to any one of the preceding items, wherein the PPG is at each occurrence independently selected from the group comprising the following groups (a1) to (a36):lerein is H, OMe or NMe2;(a4)lerein is H or Me;(a5)lereinRi is H and R2is Br; or Ri is CH3 and R2is H;lereindenotes the point of attachment of R;lereinRi is H and R2 is H;R1 is H and R2 is CH3;R1 is CH3 and R2 is H; orR1 is CH3 and R2 is CH3;lereindenotes the point of attachment of R;(a14)lereinR1 is CH, R2 is CH3 and X' is TfRi is N, R2 is CH3 and X' is Tf orR1 is CH, R2 is CeHi3 and X is Br;lereinR1 is OCH3, R2 is H and R3 is O;R1 is OCH3, R2 is H and R3 is NH;R1 is OCH3, R2 is H and R3 is S;R1 is OCH3, R2 is H and R3 is NOH;R1 is OCH3, R2 is CN and R3 is O;R1 is OCH3, R2 is CN and R3 is S;R1 is NEt2, R2 is H and R3 is O;R1 is NEt2, R2 is H and R3 is S;R1 is NEt2, R2 is CN and R3 is O; orR1 is NEt2, R2 is H and R3 is C(CN)2;lereinRi is CH3 and R2 is Et;R1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3 and R2 is Cl; orRi is CH3 and R2 is I;(a20)lereinR1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3 and R2 is CH3;orR1 is tBu and R2 is H;(a21)lerein is Zn, Pd, Cu or Ni;(a25)(a26)(a27)wherein(a) R = H and R1= NH2, NHCH3, NHEt, N(CH3)2or N(Et)2; or(b) R = Ome, NH2, NHCH3, NHEt, N(CH3)2or N(Et)2and R1= H;0(a28)(a29)wherein # denotes the point of attachment of the PPG to a photolabile self-immolative linker, a ribose 2'-O-position, a nucleobase N-atom or a nucleobase O-atom.20. The conjugate according to any one of the preceding items, wherein a nucleotide having a nucleobase with a coupled PPG comprises a structure selected from the group of:wherein * represents the coupled PPG and Rib represents the ribose moiety of the nucleotide.21. The conjugate according to any one of items 15 to 20, wherein the PPG coupled via a photolabile self-immolative linker comprises a structure selected from the group of:wherein X represents the point of attachment of the linker to the 2'-O-position or the nucleobase, and RL1, RL2, RL3and RL4are as defined in any one of the following (ia) to (id):(ia) RL1is H, RL2is H, RL3is CH3and RL4is H;(ib) RL1is H, RL2is Br, RL3is CH3and RL4is Br;(ic) RL1is H, RL2is H, RL3is NO2 and RL4is H; or(id) RL1is CH3O, RL2is H, RL3is CH3O and RL4is CH3O.22. The conjugate according to any one of the preceding items, wherein a nucleotide having a nucleobase with a coupled PPG comprises the structure:wherein NB represents the nucleobase moiety of the nucleotide, such as23. The conjugate according to any one of the preceding items, wherein a nucleotide with a PPG coupled to a ribose at the 2'-O-position comprises a structure selected from:wherein NB represents the nucleobase moiety of the nucleotide, such asThe conjugate according to any one of the preceding items, wherein the double-stranded oligonucleotide has a length of from 8 to 50 ribonucleotides, preferably a length of from 9 to 40 ribonucleotides, more preferably of from 11 to 30 ribonucleotides, even more preferably of 11 ribonucleotides or 20 to 24 ribonucleotides. The conjugate according to item 24, wherein the double-stranded oligonucleotide is blunt-ended. The conjugate according to item 24 or 25, wherein the double-stranded oligonucleotide comprises at least one diphosphate or triphosphate, preferably a triphosphate, at the 5'- end of at least the first or second strand, preferably at the 5'-end of the first strand. The conjugate according to any one of items 24 to 26, wherein the first strand or the second strand of the double-stranded oligonucleotide comprises at least one modification(s). The conjugate according to item 27, wherein the at least one modification(s) of the first strand or the second strand of the double-stranded oligonucleotide is / are selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)- modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate-morpholino-modification(s).The conjugate according to any one of items 24 to 28, wherein the at least one PPG(s) may be the same or different at each occurrence. The conjugate according to any one of items 24 to 29, wherein at least one PPG(s) is / are coupled to the 2'-O-position by a photolabile self-immolative linker. The conjugate according to item 30, wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1 , position 2, position 4, position 5, position 6, position 7, position 8, position 9, position 14, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11 , position 12, position 13, position 24, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii).32. The conjugate according to item 30 or 31 , wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1, position 5, position 6, position 7, position 8, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii).33. The conjugate according to any one of items 30 to 32, wherein one PPG is coupled at position 1 of the first strand, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of the first strand selected from position 5, position 6, position7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof.34. The conjugate according to any one of items 30 to 32, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction), one PPG is coupled at position 7 (in a 3' to 5' prime direction) of the second strand, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of the first strand selected from position 1, position 5, position6, position 7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, and any combination thereof.35. The conjugate according to item 34, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction) and one PPG is coupled at position 7 (in a 3' to 5' prime direction) of the second strand.36. The conjugate according to any one of items 24 to 35, wherein at least one PPG(s) is / are coupled to a nucleobase by a photocleavable bond or via a photolabile self- immolative linker, preferably by a photocleavable bond.37. The conjugate according to item 36, wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and / or (ii).38. The conjugate according to item 36 or 37, wherein the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in the first and / or second strand, and wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1 , 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase.39. The conjugate according to any one of items 36 to 38, wherein at least one PPG(s) is / are coupled at position 1 of the second strand and / or at position 2 of the second strand and / or at position 5 of the second strand, such as at positions 2 and 5 or positions 1, 2 and 5, or at positions 3 and 5 of the second strand.40. The conjugate according to any one of items 24, 26 to 30 and 32 to 39, wherein the double-stranded oligonucleotide has a length of from 8 to 17 ribonucleotides, atleast one diphosphate or triphosphate, preferably a triphosphate, at the 5'-end of at least the first or second strand, and further comprises a single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a di- / triphosphate, wherein the single-stranded overhang comprises at least one guanosine. The conjugate according to item 40, wherein the double-stranded oligonucleotide has a length of from 8 to 15, 8 to 12, 10 to 12, or 11 ribonucleotides, preferably 10 to 12, or 11 ribonucleotides. The conjugate according to item 40 or 41 , wherein the at least one di- / triphosphate is at the 5'-end of the strand of the double-stranded oligonucleotide which also comprises the single-stranded overhang. The conjugate according to any one of items 40 to 42, wherein the single-stranded overhang has a length of at least 3 nucleotides. The conjugate according to item 43, wherein the single-stranded overhang has a length of 3 to 15 nucleotides, 3 to 10 nucleotides, 3 to 8 nucleotides or 3 to 5 nucleotides. The conjugate according to any one of items 40 to 44, wherein the single-stranded overhang comprises at least two guanosines or at least three guanosines. The conjugate according to any one of items 40 to 45, wherein the single-stranded overhang has a length of 3 to 5 nucleotides and comprises at least three guanosines, such as three consecutive guanosines (GGG). The conjugate according to any one of items 40 to 46, wherein the single-stranded overhang comprises or consists of 5 nucleosides having the sequence adenosine- adenosine-guanosine-guanosine-guanosine (AAGGG). The conjugate according to any one of items 40 to 47, wherein the first and second strand of the double-stranded oligonucleotide form at least a blunt-end at the 5'-end of the first strand.The conjugate according to any one of items 40 to 47, wherein the double-stranded oligonucleotide comprises a further overhang, preferably a further overhang at the strand not comprising the single-stranded overhang comprising at least one guanosine. The conjugate according to item 49, wherein the further overhang is a singlestranded RNA-overhang or an overhang with a fluorophore or a lipophilic substitution. The conjugate according to any one of items 40 to 50, wherein at least the doublestranded oligonucleotide is connected with the single-stranded overhang via a linker moiety L. The conjugate according to item 51, wherein the linker moiety L is a nucleotide- linker or a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms. The conjugate according to item 51, wherein the linker moiety L is a nucleotide- linker. The conjugate according to item 51 or 53, wherein the linker moiety L is a nucleotide-linker having a length of at least 2 nucleotides. The conjugate according to any one of items 51 , 53 or 54, wherein the linker moiety L is a nucleotide-linker having a length of 2 to 10 nucleotides. The conjugate according to any one of items 51 or 53 to 55, wherein the nucleotide- linker is an adenosine-linker, preferably with at least 2 adenosines, more preferably with 2 adenosines. The conjugate according to item 51 or 52, wherein the linker moiety L is a hydrocarbon-based linker according to Formula IFormula I wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7.58. The conjugate according to any one of items 50, 52 or 57, wherein the linker-moietyL is a polyalkylene-glycol-linker according to Formula IIFormula II wherein n is an integer in the range of from 3 to 8, preferably in the range of from5 to 7.59. The conjugate according to item 58, wherein the linker moiety L is a polyalkylene- glycol-linker according to Formula II and wherein n is 6.60. The conjugate according to any one of items 40 to 59, wherein the conjugate comprises at least one modification(s) as defined in the preceding items.61 . The conjugate according to any one of items 1 to 23, wherein the at least one double-stranded oligonucleotide comprised in the conjugate comprises a structure according to Formula III, Formula IV or Formula V:RNA1 - BL1 - RNA3 RNA1 RNA3RNA2 RNA4 RNA2 - BL2 -RNA4Formula III Formula IVRNA1 - BL1 - RNA3RNA2 - BL2 - RNA4Formula V whereinRNA1 represents the first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA2 represents the second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the first strand via complementary base pairing;RNA3 represents a third strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA4 represents a fourth strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the third strand via complementary base pairing;BL1 , if present, represents a bivalent linker that covalently bonds the 3'-end of RNA1 to the 3'- or 5'-end of RNA3;BL2, if present, represents a bivalent linker that covalently bonds the 5-end of RNA2 to the respective 5'- or 3'-end of RNA4; whereinRNA1 and RNA2, as well as RNA3 and RNA4, each have no overhang of the 5’- terminal nucleotide residues, and wherein RNA1 and RNA2, as well as RNA3 and RNA4, each can comprise an overhang of the 3’-terminal nucleotide residues of not more than five nucleotides, preferably of not more than four or three nucleotides, more preferably of not more than two nucleotides, even more preferably of not more than one nucleotide, especially no overhang of the 3’-terminal nucleotide residues.62. The conjugate according to item 61, wherein the linker BL1 and BL2, each independent from each other, is selected from the group of a nucleotide-linker as defined in the preceding items, a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms as defined in the preceding items, a phosphodiester linker (OPO(OH)O-), and any combination thereof.63. The conjugate according to item 61 or 62, wherein the sequence of RNA1 is identical to the sequence of RNA3.64. The conjugate according to any one of items 61 to 63, wherein the sequence of RNA2 is identical to the sequence of RNA4.65. The conjugate according to any one of items 61 to 64, wherein each of RNA1 , RNA2, RNA3 and RNA4 has a length of from 9 to 40 nucleotides, preferably of from 11 to 30 nucleotides, more preferably of from 20 to 24 nucleotides.The conjugate according to any one of items 61 to 64, wherein each of RNA1 , RNA2, RNA3 and RNA4 has a length of from 8 to 11 nucleotides, preferably 11 nucleotides. The conjugate according to any one of items 61 to 66, wherein the conjugate comprises a structure according to Formula III, wherein BL1 bonds the 3'-end of RNA1 to the 3'-end of RNA3, and wherein RNA1 and RNA3 each have, at their 5'- end, a diphosphate or triphosphate, preferably a triphosphate. 68. The conjugate according to any one of items 61 to 66, wherein the conjugate comprises a structure according to Formula IV, wherein BL2 bonds the 5'-end of RNA2 to the 5'-end of RNA4, and wherein RNA1 and RNA3 each have, at their 5'- end, a diphosphate or triphosphate, preferably a triphosphate. The conjugate according to any one of items 61 to 66, wherein the conjugate comprises a structure according to Formula V, wherein BL1 bonds the 3'-end of RNA1 to the 3'-end of RNA3, BL2 bonds the 5'-end of RNA2 to the 5'-end of RNA4, and wherein RNA1 and RNA3 each have, at their 5'-end, a diphosphate or triphosphate, preferably a triphosphate. The conjugate according to any one of items 61 to 66, wherein the conjugate comprises a structure according to Formula V, wherein BL1 bonds the 3'-end of RNA1 to the 5'-end of RNA3 and BL2 bonds the 5'-end of RNA2 to the 3'-end of RNA4, wherein the strands of the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 are not complementary, and wherein RNA1 and RNA4 each have, at their 5'-end, a diphosphate or triphosphate, preferably a triphosphate. The conjugate according to any one of items 61 to 70, wherein the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 may have the same or a different length. The conjugate according to any one of items 61 to 71 , wherein the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 each are blunt-ended double-stranded RNA. The conjugate according to any one of items 61 to 72, wherein at least one of RNA1, RNA2, RNA3 and RNA4 comprises at least one modification(s).74. The conjugate according to item 72, wherein the at least one modification(s) of at least one of RNA1 , RNA2, RNA3 and RNA4 is / are selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2 '-O- methyl-modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group- modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate-morpholino-modification(s).75. The conjugate according to any one of items 61 to 74, wherein, in addition to RNA1 and / or RNA2, at least one of RNA3 and RNA4 comprises at least one PPG(s).76. The conjugate according to any one of items 61 to 75, wherein the at least one PPG(s) in RNA1 , RNA2, RNA3 and / or RNA4 may be the same or different at each occurrence.77. The conjugate according to any one of items 61 to 76, wherein the at least one PPG(s) comprised in at least one of RNA1 , RNA2, RNA3 and / or RNA4 is / are coupled to 2'-O-position via a photolabile or self-immolative linker.78. The conjugate according to item 77, wherein the conjugate is a conjugate according to any one of items 67 to 69, and wherein the at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1 , position 5, position 6, position 7, position 8, position 9, and any combination thereof;(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11, and any combination thereof; or(iii) two or more PPGs are coupled to RNA1, RNA2, RNA3 and RNA4 at positions selected from (i) and (ii).79. The conjugate according to item 78, wherein one PPG is coupled at position 1 of RNA1 and / or RNA3, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of RNA1 and / or RNA3 selected from position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11, and any combination thereof.80. The conjugate according to item 78, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction), one PPG is coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and RNA4, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1, position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, position 11 and any combination thereof.81. The conjugate according to item 80, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction) and one PPG is coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA4.82. The conjugate according to item 77, wherein the conjugate is a conjugate according to item 70, and wherein at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1 , position 5, position 6, position 7, position 8, position 9, and any combination thereof;(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11, and any combination thereof; or(iii) two or more PPGs are coupled to RNA3 and / or RNA4 at positions selected from (i) and (ii).83. The conjugate according to item 82, wherein one PPG is coupled at position 1 of RNA1 and / or RNA4, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of RNA1 and / or RNA4 selected from position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11, and any combination thereof.84. The conjugate according to item 82 or 83, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction), one PPG is coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and RNA3, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1, position 5, position 6, position 7, position 8, position 9, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, position 11, and any combination thereof.85. The conjugate according to item 84, wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction) and one PPG is coupled at position 7 (in a 3' to 5' prime direction) of RNA2 and / or RNA3.86. The conjugate according to any one of items 61 to 76 and 78 to 85, wherein at least one PPG(s) is / are coupled to a nucleobase by a photocleavable bond or via a photolabile self-immolative linker, preferably by a photocleavable bond.87. The conjugate according to item 86, wherein the conjugate is a conjugate according to any one of items 67 to 69, and wherein the at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA3 selected from position 1 , position2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA4, in a 3' to 5' prime direction, selected from position 1 , position 2 position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof.88. The conjugate according to item 86 or 87, wherein the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in one or more of RNA1, RNA2, RNA3 and RNA4, and wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1, 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase.89. The conjugate according to any one of items 86 to 88, wherein at least one PPG(s) is / are coupled at position 1 and / or at position 2 and / or at position 5, such as at positions 2 and 5 or positions 1, 2 and 5, of RNA2 and / or RNA4, or at positions 3 and 5 of RNA2 and / or RNA4.90. The conjugate according to item 86, wherein the conjugate is a conjugate according to item 70, and wherein the at least one PPG(s) is / are coupled at:(i) a position of RNA1 and / or RNA4 selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; and / or(ii) a position of RNA2 and / or RNA3, in a 3' to 5' prime direction, selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof.91. The conjugate according to item 90, wherein the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in one or more of RNA1, RNA2, RNA3 and RNA4, and wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1 , 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase.92. The conjugate according to item 90 or 91, wherein at least one PPG(s) is / are coupled at position 1 and / or at position 2 and / or at position 5, such as at positions 2 and 5 or positions 1, 2 and 5, of RNA2 and / or RNA3, or at positions 3 and 5 of RNA2 and / or RNA3.93. The conjugate according to any one of items 1 to 23, wherein the conjugate has a structure according to Formula VI:Formula VI wherein sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of a mRNA strand;L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms; and dsO represents a double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and (ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand.The conjugate according to item 93, wherein the conjugate is a nucleic acidconstruct, preferably an engineered nucleic acid-construct. The conjugate according to item 93 or 94, wherein the dsO is as defined in any one of items 24 to 60. The conjugate according to any of items 93 to 95, wherein L3 connects the 3'-end of the first strand of the dsO or the 5'-end of the second strand of the dsO with the sN. The conjugate according to any of items 93 to 95, wherein L3 is absent, and the sN is directly connected to the 3'-end of the first strand of the dsO or to the 5'-end of the second strand of the dsO, preferably to the 3'-end of the first strand. The conjugate according to any of items 93 to 97, wherein sN is complementary to at least one nucleic acid sequence comprised in a mRNA. The conjugate according to any of items 93 to 98, wherein the sN is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of a mRNA. The conjugate according to item 99, wherein the non-coding nucleic acid sequence of the mRNA is or comprises a 3'-untranslated region (UTR)-nucleic acid sequence or a 5'-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail. The conjugate according to item 99 or 100, wherein the sN capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'-untranslated region is complementary to at least one nucleic acid sequence comprised in the mRNA strand. The conjugate according to any one of items 1 to 23, wherein the conjugate comprises a structure according to Formula VII:Formula VII wherein mRNA is a mRNA-strand;(sN-L3-dsO) represents a unit comprising (a) to (c), wherein(a) sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand;(b) L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms;(c) dsO represents a double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and(ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand; and m is an integer in the range of from 1 to 4.103. The conjugate according to item 102, wherein the conjugate is a nucleic acidconstruct, preferably an engineered nucleic acid-construct.104. The conjugate according to item 102 or 103, wherein m is 2, 3 or 4, preferably m is 4.105. The conjugate according to any of items 102 to 104, wherein in each unit (sN-L3- dsO) each of the individual elements sN, L3 and dsO is independently selected.106. The conjugate according to any one of items 102 to 105, wherein each of the elements sN, L3 and dsO, in each unit (sN-L3-dsO), is the same or different at each occurrence.The conjugate according to any one of items 102 to 106, wherein sN is complementary to at least one nucleic acid sequence comprised in the mRNA strand. The conjugate according to any one of items 102 to 107, wherein the mRNA-strand comprises a coding sequence of a protein, a peptide or an antigen, preferably an antigen that is able to induce an immune response or a cancer antigen. The conjugate according to any one of items 102 to 108, wherein sN is capable of hybridizing to at least one nucleic acid sequence located in a non-coding nucleic acid sequence of the mRNA-strand. The conjugate according to item 109, wherein the non-coding nucleic acid sequence of the mRNA-strand is or comprises a 3'-untranslated region (UTR)- nucleic acid sequence or a 5'-untranslated region (UTR)-nucleic acid sequence or a poly-A-tail. The conjugate according to any one of items 102 to 110, wherein the mRNA-strand comprises a 3'-untranslated region (UTR)-nucleic acid sequence, and wherein the sN is capable of hybridizing to at least one nucleic acid sequence of the mRNA- strand comprised in said 3'-untranslated region (UTR)-nucleic acid sequence. The conjugate according to any one of items 102 to 110, wherein the mRNA-strand comprises a 5'-untranslated region (UTR)-nucleic acid sequence, and wherein the sN is capable of hybridizing to at least one nucleic acid sequence of the mRNA- strand comprised in said 5'-untranslated region (UTR)-nucleic acid sequence. The conjugate according to item 111 or 112, wherein the sN is capable of hybridizing to the at least one nucleic acid sequence comprised in the 3'- or 5'- untranslated region (UTR)-nucleic acid sequence of the mRNA-strand via Watson- Crick-base-pairing. The conjugate according to any one of items 102 to 113, wherein the dsO further comprises an overhang at the first or second strand.The conjugate according to item 114, wherein the overhang is a RNA-overhang or an overhang with a fluorophore sterol, vitamin or a lipophilic substitution. The conjugate according to any one of items 102 to 115, wherein the dsO has a length in the range of from 9 to 40 nucleotides, preferably of from 11 to 30 nucleotides, more preferably of from 20 to 24 nucleotides. The conjugate according to any one of items 102 to 115, wherein the dsO has a length in the range of from 8 to 11 nucleotides, preferably 11 nucleotides. The conjugate according to any one of items 102 to 118, wherein the mRNA-strand comprises at least one modification(s). The conjugate according to item 118, wherein the at least one modification(s) in the mRNA-strand is / are pseudouridine-modification(s), preferably N1-methyl- pseudouridine-modification(s), methoxyuridine-modification(s), preferably 5- methoxyuridine-modification(s), methylcytosine-modification(s), preferably 5- methylcytosine-modification(s), methyl-adenosine-modification(s), preferably N6- methyl-adenosine-modification(s). The conjugate according to item 118 or 119, wherein the at least one modification(s) of the mRNA-strand is / are pseudo-uridine-modification(s). The conjugate according to any one of items 102 or 120, wherein the first and / or second strand of the dsO comprise(s) at least one modification(s). The conjugate according to item 121, wherein the first strand comprises at least one modification(s) at its 3'-end. The conjugate according to item 121 or 122, wherein the second strand comprises at least one modification(s) at its 5'-end. The conjugate according to any one of items 121 to 123, wherein the at least one modification(s) of the first and / or second strand of the dsO is / are selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O-methoxyethyl-modification(s), fluoro- group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid(LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate-morpholino-modification(s). The conjugate according to any one of items 121 to 124, wherein the second strand comprises at least one phosphorothioate-modification(s). The conjugate according to any one of items 102 to 125, wherein L3 comprises 8 to 16, 10 to 14 or 12 main chain atoms. The conjugate according to any one of items 102 to 126, wherein the main chain atoms are carbon atoms that are optionally replaced by one or more heteroatoms selected from the group consisting of N, O and S. The conjugate according to any one of items 96 and 102 to 127, wherein L3 is a linker as defined in items 57 to 59. The conjugate according to any one of items 102 to 128, wherein L3 is is bonded to the 3'-end of the first strand or the 5'-end of the second strand of the dsO. The conjugate according to any one of items 99 and 102 to 129, wherein sN has a length in the range of from 5 to 15, such as 8 to 13 nucleotides. The conjugate according to item 102 or 104, wherein each unit (sN-L3-dsO) independently comprises or consists of a structure of Formula VI according to any one of items 93 to 101. The conjugate according to any one of the preceding items for use as a medicament. The conjugate according to any one of the preceding items for use as a vaccine, preferably the conjugate according to any one of items 102 to 131. The conjugate for use according to item 132 or 133, comprising directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide at the target site, wherein the activated or released double-stranded oligonucleotide is capable of inducing apoptosis of a tumor cell, of inducing an anti-viral immune response, of inducingbinding to and / or activation of cytosolic helicase retinoic acid-inducible gene I (RIG- I), and / or of inducing IFN production, preferably of inducing type I IFN production. The conjugate according to items 93 to 101 for use an adjuvant, preferably as vaccine or immune adjuvant, more preferably as mRNA vaccine adjuvant. Adjuvant comprising a conjugate comprising or consisting of a structure according to Formula VI. mRNA vaccine comprising or consisting of one or more conjugate(s) comprising the structure according to Formula VII. Pharmaceutical composition comprising the conjugate according to any one of items 1 to 101 and, optionally, at least one pharmaceutically acceptable carrier, excipient or delivery agent. Drug delivery system comprising the conjugate according to any one of items 1 to 101 , characterized in that the conjugate is a caged drug comprising the doublestranded oligonucleotide as therapeutic agent that is rendered biologically inactive by being coupled to at least one PPG(s); wherein said double-stranded oligonucleotide is capable of being activated and / or released in response to a predetermined wavelength and / or intensity of light which removes the at least one coupled PPG(s) tracelessly. The drug delivery system according to item 139, which further comprises a marker. The drug delivery system according to item 140, wherein the marker is luminescent. The conjugate according to any one of items 1 to 101 , the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 for use in a method of treatment or prevention of a disease, comprising: administering the conjugate or system to a patient, localising the conjugate or system at the target site, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site.The conjugate, composition or system for use according to item 142, wherein the conjugate, composition or system is administered orally or parenterally to the patient. The conjugate, composition or system for use according to item 143, wherein the conjugate , composition or system is administered parenterally by intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing. The conjugate, composition or system for use according to any one of items 142 to144, wherein the disease is cancer or an infection, preferably a virus infection. The conjugate, composition or system for use according to any one of items 142 to145, 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 tumor (PNET)), and combinations thereof. The conjugate, composition or system for use according to any one of items 142 to 145, wherein the disease is a hematological tumor or a solid tumor. The conjugate according to any one of items 1 to 101 , the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 for use in a method of infiltrating immune cells, preferably T cells. The conjugate according to any one of items 1 to 101 , the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 for use in a method of turning cold into hot tumors.Use of the conjugate according to any one of items 102 to 131 or the drug delivery system according to any one of items 139 to 141 , which comprises the conjugate according to any one of items 102 to 131 , as a vaccine. Use of the conjugate according to any one of items 1 to 101 as an adjuvant. Use of the conjugate according to any one of items 1 to 101 in the manufacture of a medicament for the treatment or prevention of a disease. The use according to item 152, wherein the disease is cancer or an infection, preferably a virus infection. The use according to item 152 or 153, 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 tumor (PNET)), and combinations thereof. The use according to item 152 or 153, wherein the disease is a hematological tumor or a solid tumor. The conjugate according to any one of items 1 to 101, the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 for use in a method of inducing an immune response. The conjugate according to any one of items 1 to 131, wherein the conjugate is an antigen recognizing conjugate or part of an antigen recognizing conjugate or is comprised in an antigen recognizing conjugate.The conjugate according to item 157, wherein the antigen recognizing conjugate 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. A method of treating a disease, comprising the step of administering a therapeutically effective amount of the conjugate according to any one of items 1 to 101 , the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site. The method of treating according to item 159, wherein the disease is cancer or an infection, preferably a virus infection. The method of treating according to item 160, 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 tumor (PNET)), and combinations thereof. The method of treating according to item 160, wherein the disease is a hematological tumor or a solid tumor. The method of treating according to any one of items 159 to 162, wherein the conjugate is administered in a multimerized form. The method of treating according to any one of items 159 to 163, wherein the therapeutically effective amount of the conjugate is administered to the subject in need thereof orally or parenterally.The method of item 164, wherein the therapeutically effective amount of the conjugate is administered to the subject in need thereof parenterally by intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing. A method of inducing an immune response, comprising the step of administering a therapeutically effective amount of the conjugate according to any one of items 1 to 101 , the pharmaceutical composition of item 138 or the drug delivery system according to any one of items 139 to 141 to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged double-stranded oligonucleotide as immune stimulant at the target site. The method according to item 166, wherein the immune response is induced by stimulating retinoic acid-inducible gene I (RIG-I). The method according to any one of items 166 or 167, wherein the conjugate has or comprises an immune adjuvant function. The method according to any one of items 166 to 168, wherein the conjugate is a conjugate according to any one of items 102 to 131 comprising:(a) at least one unit (sN-L3-dsO) capable of functioning as RIG-l-ligand;(b) at least two units (sN-L3-dsO) capable of functioning as RIG-l-ligands;(c) at least three unit (sN-L3-dsO) capable of functioning as RIG-l-ligands; or(d) at least four unit (sN-L3-dsO) capable of functioning as RIG-l-ligands. The method according to any one of items 166 to 169, wherein the therapeutically effective amount of the conjugate is administered to the subject in need thereof orally or parenterally. The method of item 170, wherein the therapeutically effective amount of the conjugate is administered to the subject in need thereof parenterally wherein the therapeutically effective amount of the conjugate is administered to the subject in need thereof parenterally by intravenous injection, intratumoral injection,intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing.172. A kit for use in medicine comprising the conjugate according to any one of items 1 to 131.173. The kit according to item 172, 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.

[0332] 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.

[0333] As used herein, “a” and “an” refer to not only a single individual, but also a group or species of entities unless otherwise noted.

[0334] 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).

[0335] 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.

[0336] 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.

[0337] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. 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.EXAMPLESMaterials & methodsCells

[0338] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation from whole blood of healthy volunteers and from buffy coats. PBMCs were plated in RPMI 1640, supplemented with 10% fetal calf serum (FCS), 100 pg / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate and 0,1 mM MEM non-essential amino acids (all from Thermo Fisher Scientific / Gibco) and incubated in a humidified incubator at 37°C and 5% CO2 at a density of 4x106cells / mL.

[0339] CD14 positive monocytes where isolated from prior isolated PBMCs using the CD14+Monocyte Isolation Kit, human from Miltenyi Biotec according to the instructions. Monocytes were plated in RPMI 1640, supplemented with 10% fetal calf serum (FCS), 100 pg / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate and 0,1 mM MEM non-essential amino acids (all from Thermo Fisher Scientific / Gibco) and incubated in a humidified incubator at 37°C and 5% CO2 at a density of 1x106cells / mL.

[0340] THP1 Dual wt cells (Invivogen) were cultured in RPMI 1640, supplemented with 10% fetal calf serum (FCS), 100 lll / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate and 0,1 mM MEM non-essential amino acids (all from Thermo Fisher Scientific / Gibco), in a humidified incubator at 37°C and 5% CO2 and plated at a density of 8x105cells / mL.

[0341] HeLa (CCL-2; ATCC), A375 and A549 cells were cultured in DMEM high glucose full medium, supplemented with 10% fetal calf serum (FCS), 100 pg / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate and 0,1 mM MEM non-essential amino acids (all from Thermo Fisher Scientific), and seeded one day before transfection at a density of 2x105cells / mL. If not else specified they were incubated in a humidified incubator at 37°C and 5% CO2. A375 cells were kindly provided by Michael Holzel (University Hospital Bonn, Germany) and A549 cells were kindly provided by Christine Goffinet (Charite Berlin, Germany).

[0342] HcMel3 cells were generated as previously described (see, e.g. Landsberg et al, 2012) and cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum (FCS), 100 pg / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate and 0,1 mM MEM non-essential amino acids (all from Thermo Fisher Scientific), and seeded one day before transfection at a density of 2x105cells / mL. If not else specified they were incubated in a humidified incubator at 37°C and 5% CO2.

[0343] Primary ccRCC (clear cell renal cell carcinoma) cells were isolated as previously described (see, e.g. Esser et al, 2022) and cultivated in DMEM / F12 medium, supplemented with 5% FCS (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 10 ng / mL hrEGF (R&D Systems), 10 ng / mL FGF-basic (PeproTech), 4 pg / ml Heparin (Thermo Scientific Chemicals), 1 x B27-supplement without vitamin A (Thermo Fisher Scientific), 1 x Lipid Mixture 1 (Sigma- Aldrich), 1- mM N-Acetyl-Cysteine (Thermo Fisher Scientific), 1 x GlutaMAX (Thermo Fisher Scientific), 1 x MEM non-essential amino acids (Thermo Fisher Scientific) and 10-mM HEPES (GE Healthcare, UK), in a humidified incubator at 37°C and 5% CO2. One day before transfection ccRCC cells were seeded in 96-well plates at a density of 2x105cells / mL.

[0344] Cell lines were verified to be mycoplasma negative by using a mycoplasma-specific PCR approach at regular intervals.Lipofection based transfection

[0345] Transfection was performed with the indicated concentrations of oligonucleotides using Lipofectamine2000® (Invitrogen) according to the instructions. 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.

[0346] Transfection of mRNA samples was performed with the indicated concentrations of oligonucleotides using Lipofectamine™ MessengerMax™ (Invitrogen) according to the instructions. Briefly, oligonucleotides and Lipofectamine™ MessengerMax™ dilutions were prepared in OptiMEM (Gibco) and incubated for 10 min. Afterward, the RNA / OptiMEM and Lipofectamin / OptiMEM solutions were mixed in equal volume and incubated for additional 5 min at RT. 10 pL of the mixture was added to each well resulting in the indicated concentration.In vivo-jetPEI based transfection

[0347] Cells were transfected with RNA complexed with in vivo-jetPEI (Polyplus) according to the manufactures protocol. Briefly, the oligonucleotides and in vivo-jetPEI dilutions were prepared in 5% Glucose (Polyplus), using 0.16 pL in vivo-jetPEI per pg RNA. Afterwards, the RNA / Glucose and in vivo-jetPEI / Glucose solutions were mixed in equal volumes and incubated for additional 15 min at RT. 25 pL of the mixture was added to each well resulting in the indicated concentrations.Invivofectamine 3.0 based transfection

[0348] Cells were transfected with RNA complexed with Invivofectamine 3.0 (Thermo Fischer) according to the manufactures protocol. Briefly, the oligonucleotides were incubated with the Invivofectamine 3.0 reagent (1.5 pL per pg RNA) for 30 minutes at 50°C in complexation buffer. Following dilution with PBS (Gibco), 25 pL of the mixture was added to each well resulting in the indicated concentrations.Uncaging - Activation of double-stranded oligonucleotide

[0349] Photocaged double-stranded RNA conjugates were uncaged directly after transfection by irradiating transfected cells with light-emitting diodes (LED, 365 nm, 2 mW, indicated time).

[0350] Unless otherwise specified, cells were stimulated for 18 hours.Readout of RIG-I stimulationELISA

[0351] The level of secreted of IFN-a (Thermo Fisher Scientific) as result of RIG-I stimulation by the uncaged conjugates was quantified in cell-free supernatants by ELISA according to the manufacturer's instructions.

[0352] The level of secreted IFN-a (Thermo Fisher Scientific) and CXCL-10 (BD Biosciences) as result of RIG-I stimulation by the uncaged conjugates was quantified in cell-free supernatants by ELISA according to the manufacturer's instructions.Luciferase Assay

[0353] Activity of the THP1 dual ISG54 minimal promoter in conjunction with five IFN- stimulated response elements was measured by quantifying the Lucia luciferase reporter gene expression. Luciferase assay was performed 18 h after stimulation. Cell- free supernatants were mixed in a 1 :1 ratio with luciferase substrate (1 pg / mL coelenterazine in 100 mM Tris pH 7.4, 300 mM Sodium Ascorbate). The luminescence was measured using an EnVision 2104 Multilabel Reader (PerkinElmer).Annexin V / 7-AAD staining and FACS analysis

[0354] Following 72 hours of incubation cells were harvested and washed with 100 pL PBS (phosphate buffered saline, Thermo Fisher Scientific / Gibco). Then, cells were incubated with Annexin V-Alexa 647 (diluted 1 :30; BioLegend) in Annexin binding buffer (10 mM HEPES, pH 7,4; 140 mM NaCI; 2,5 mM CaCI2) for 15 minutes at room temperature (RT) in the dark. Subsequently, cells were resuspended in 200 pL Annexin binding buffer, containing 1 ,25 pg / mL 7-AAD (7-Aminoactinomycin D, Enzo Life Sciences). FACS measurement was performed immediately after the staining process. Fluorescence intensities were measured with the Attune NxT Flow Cytometer (Thermo Fischer) and results analyzed using FlowJo software.

[0355] Table 3: Sequences used in the ExamplesResults

[0356] In the following examples different conjugates according to the present invention or model molecules mimicking a photocage substituent have been used. The tested conjugates and model molecules comprised dsRNA as double-stranded oligonucleotide. The dsRNA was either linear (see, e.g. Figure 1 and Examples 1 and 2) or comprised dsRNA with a guanosine containing overhang (see, e.g. Fig. 8A and Example 3). In addition, the dsRNAs varied in both sequence and length. Photocages(or a model substituent) were coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand of the dsRNAs. The positions to which photocages were coupled to in the first or second strand of the tested dsRNAs varied, including position 1 of the first strand and positions 6 and 7 of the second strand. Number of photocages present in the dsRNAs was also varied.

[0357] The inventors of the present invention could demonstrate that RIG-I mediated IFN- expression in reporter cell lines could be light-controlled using conjugates according to the invention, thus providing a unique spatial and temporal control of targeted activation of RIG-I and avoidance of unwanted systemic side-effects, such as, for example, an uncontrolled overreaction of the innate immune system. Most importantly, the inventors could demonstrate that this advantageous light-controlled activation is neither particularly dependent on sequence of the double-stranded oligonucleotide nor on number or specific position of the photocage. However, the number of photocages present may have an influence on light-control.

[0358] The inventors of the present invention have thus surprisingly found conjugates which satisfies the long-felt need of temporally and spatially controlled targeted activation of RIG-I, which can be of tremendous benefit in RIG-I ligand-based immunotherapy as well as in the context of making therapy-resistant tumors available for treatment or of vaccination, in particular mRNA-based vaccination as descried above.Example 1 :

[0359] By the results shown in Figure 6, the inventors demonstrate that a single photocage at the 2’-O-position of the first nucleotide of the first strand of the double-stranded oligonucleotide in a conjugate according to the invention provides light-controlled targeted activation of RIG-I as indicated by the high IFN expression levels observed in human peripheral blood mononuclear cells (PBMCs) after uncaging (see Figure 1). PBMCs were transfected with negative control RNA (neg Ctrl), photocaged RIG-I ligand RNA (N1-2'-O-photocage; dsRNA with PPG coupled to ribose at the 2'-O-position of the first nucleotide (N1 position) of the sense strand) or RIG-I ligand control RNA (3p- dsRNA) using Lipofectamine™2000 (Invitrogen) and 1.28 ng / mL of each sample RNA (0.16 ng / well). After transfection, a part of the transfected cells (samples indicated with +hv) was irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes), while the other part was not irradiated (samples indicated -hv). After 18 hours, the supernatants were removed from the irradiated and non-irradiated transfected cells and IFN-a production was determined using ELISA. The results shown in Figure 6 represent at least three experiments and mean ± SEM is shown.

[0360] No IFN-a production was observed for the negative control (neg Ctrl) and the nonirradiated cells with photocaged RNA (N1-2'-O-photocage, -hv).

[0361] Transfection with RIG-I ligand control RNA (3p-dsRNA) resulted in similar levels of IFN-a production for the non-irradiated and irradiated samples.

[0362] Irradiation of the cells with photocaged RNA (N1-2'-O-photocage, + hv) elicited a comparable level of IFN-a production as the positive control RIG-I ligand control RNA.

[0363] It was further observed that RIG-I induced IFN production after activation of RIG-I is time-dependent and that a conjugate according to the present invention (photocaged dsRNA) only acts after activation with light as RIG-I ligand (data not shown).

[0364] This demonstrates that the photocaged dsRNA acts as RIG-I ligand after activation with light and that the 2'-O-photocage at position N1 effectively inactivates (masks) essential molecular motifs needed for RIG-I activation, such as, e.g., RIG-l-mediated IFN responses. Thus, the results presented in this figure provide general proof of concept that a conjugate according to the invention masks biological activity of the double-stranded oligonucleotide, and that the activity of the double-stranded oligonucleotide can be restored on demand in a spatiotemporally controlled manner by irradiation with light.Example 2:

[0365] By the results shown in Figure 7, the inventors further demonstrate that photocontrollable activation of RIG-I as indicated by IFN-expression levels is also possible with conjugates according to the invention, which have a single PPG coupled to different nucleotides in the second strand (antisense strand) of the double-stranded oligonucleotide, and that the presence of two PPGs in the second strand at these different nucleotide positions led to comparable light-controlled activation.

[0366] As in Example 1 above, peripheral blood mononuclear cells (PBMCs) were transfected with negative control RNA (neg Ctrl), RIG-I ligand control RNA (3p-dsRNA) and the following conjugate sample RNAs using Lipofectamine™2000 (Invitrogen): 3p- dsRNA + N3.6 2’O-photocage, 3p-dsRNA + N3.7 2’O-photocage and 3p-dsRNA + N3.6+N3.7 2’O-photocage. These conjugate sample RNAs have a 2'-O-photocage in the second strand (antisense strand “N3”) at the following position(s), counted from the 5'-end of the first strand (i.e. 3' to 5' prime direction of the second strand): nucleotide 6 (N3.6), nucleotide 7 (N3.7), and nucleotides 6 and 7 (N3.6 + N3.7). 800 ng / mL of each sample RNA was used for transfection. A part of the transfected cells was irradiated (samples indicated +hv) with light-emitting diodes (365 nm, 2mW, 10 minutes), while the other part was not irradiated (samples indicated -hv). After 18 hours, the supernatants were removed from the irradiated and non-irradiated transfected cellsand IFN-a production was determined using ELISA. The results shown in Figure 7 represent at least three experiments and mean ± SEM is shown.

[0367] Each irradiated conjugate sample “N3.6” (most left panel in grey), “N3.7” (second panel from left in light grey), and “N6,N7” (second panel from right in grey) induced levels of IFN-expression comparable to that of the positive control, i.e the known RIG- I ligand 3p-dsRNA (dark grey bar). Basically, no relevant activation was observed for the negative control and non-irradiated photocaged samples. However, the masking effect was higher when two photocages were present.

[0368] Thus, light-controlled targeted activation of RIG-I is enabled by conjugates according to the invention having at least one PPG coupled to a ribose 2’-O-position in the second strand of the double-stranded oligonucleotide.

[0369] The presence of more than one PPG can increase the masking effect, and thus light control.

[0370] The results demonstrate that photocontrollable activation of RIG-I is basically independent of position of the coupled photocage(s).All in all, Example 2 provides confirmation and general proof of concept that a conjugate according to the invention with at least one PPG present in the second strand successfully masks biological activity of the double-stranded oligonucleotide, and that the activity of the double-stranded oligonucleotide can be restored on demand in a spatiotemporally controlled manner by irradiation with light.Example 3:Example 3 shows that controlled targeted activation of RIG-I is independent of RNA nucleotide sequence and that different linkers can be used for connecting a guanosinecontaining overhang. Each of the two dsRNAs “GFP2-AAGGG” and “GI2-HEG-GGG”, which differ in nucleotide sequence of 11 bp, but both comprise a triphosphate at the 5'- end of the first (sense) strand, and a single-stranded guanosine containing overhang at the 3'-end of the first strand, were applied to THP1 Dual™ wt cells (InvivoGen). In addition, “GFP2-AAGGG” and “GI2-HEG-GGG”, each modified by 2'-O-methyl-group-modifactions at positions N3.6 and N3.7, in a 3' to 5' prime direction, of the second (anti-sense) strand, and a negative control RNA were applied. Cells were transfected with 800 ng / mL of each sample RNA using Lipofectamine™2000 (Invitrogen). 18 hours after transfection, supernatants were collected to determine the levels of Lucia luciferase induction by the interferone-stimulated response element (ISRE) reporter, which surrogates for the level of IFN production, stimulated by exposure to the sample RNAs using the Luciferase assay. The results are shown in Figure 8B and 8C; they represent at least two experiments and mean ± SEM is shown.

[0371] Both, “GFP2-AAGGG” and “GI2-HEG-GGG”, despite their different sequence and linker moieties had a very high IFN-stimulating activity (see, black bar in Figure 8B and 80). Thus, the results demonstrate and prove that the ability to induce IFN expression via activation of RIG-I does neither depend on precise sequence of nucleotides in the double-stranded oligonucleotide nor on a specific linker moiety as long as these elements have the key structural features as described and defined herein - in this example, a certain length, 5’-triphosphate and 3’-guanosine-containing overhang with adenosine or hydrocarbon-based (hexaethylene glycol) linker.

[0372] Modification of nucleotides N3.6 and N3.7 in the second (antisense) strand of “GFP2-AAGGG” and “GI2-HEG-GGG” by 2'-O-methyl-group-modifactions inactivated (masked) the very high IFN-stimulating activity (see, light grey bar in Figure 8B and 80). In other words, the results demonstrate and provide proof of concept that the ability to induce IFN expression via activation of RIG-I can be masked by modification of the 2'-O-position in nucleotides of the second strand of the double-stranded oligonucleotide as described and defined herein - in this example, the 2'-O-position of nucleotides N3.6 and N3.7 in the second (antisense) strand.This demonstrates that controlled target activation of RIG-I does not depend an RNA nucleotide sequence and is enabled for conjugates comprising a double-stranded oligonucleotide with a rather short / minimal length and a guanosine-containing overhang as described and defined herein.Example 4:

[0373] In this example, whose results are shown in Figure 9, the inventors show that light- controlled targeted activation of RIG-I is enabled by a conjugate according to the invention that has at least one PPG coupled to a nucleobase of at least one nucleotide (cf. exemplary embodiment in Figure 9A) in the second (anti-sense) strand of a doublestranded nucleotide capable of binding to and activating RIG- 1.In Figure 9B peripheral blood mononuclear cells (PBMCs) were transfected with 800 ng / mL of each of: negative control RNA (neg Ctrl), nucleobase-photocaged RNA (3p- ds RNA + N3.1 -basecage; dsRNA with a PPG coupled to the nucleobase of the first nucleotide (N3.1 position) of the second (anti-sense) strand) and RIG-I ligand control RNA (3p-dsRNA) using Lipofectamine™2000 (Invitrogen). After transfection, a part of the transfected cells (samples indicated with +hv) was irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes), while the other part was not irradiated (samples indicated - hv). After 18 hours, the supernatants were removed from the irradiated and non-irradiated transfected cells and IFN-a production was determined using ELISA. The results shown in Figure 9B represent at least three experiments and mean ± SEM is shown.In Figures 9C, 9D and 9E CD14 positive monocytes were transfected with 800 ng / mL of each of: negative control RNA (neg Ctrl), a nucleobase-photocaged RNA and RIG-I ligand control RNA (ds GI2 II) using Lipofectamine™2000 (Invitrogen). The nucleobase-photocaged RNAs tested were as follows: C. GI2 II + N3.1 ,N3.2-basecage; dsRNA with a PPG coupled to the nucleobase of the first and second nucleotide (N3.1 and N3.2 position) of the second (anti-sense) strand; D. GI2 II + N3.2,N3.5-basecage; dsRNA with a PPG coupled to the nucleobase of the second and fifth nucleotide (N3.2 and N3.5 position) of the second (anti-sense) strand; and E. GI2 II + N3.1 ,N3.2,N3.5-basecage; dsRNA with a PPG coupled to the nucleobase of the first, second and fifth nucleotide (N3.1 and N3.2 and N3.5 position) of the second (anti-sense) strand. After transfection, a part of the transfected cells (samples indicated with +hv) was irradiated with light-emitting diodes (365 nm, 2mW, 10 minutes), while the other part was not irradiated (samples indicated - hv). After 18 hours, the supernatants were removed from the irradiated and non-irradiated transfected cells and IFN-a production was determined using ELISA. The results shown in Figure 9C-9E represent at least two experiments and mean ± SEM is shown.

[0374] Irradiation of transfected cells with nucleobase-photocaged RNA (3p-ds RNA + N3.1 -basecage, +hv) elicited a comparable level of IFN-a production (light grey bar, Figure 9B) as the positive control RNA (RIG-I ligand "3p-dsRNA", Figure 9B, black bar).

[0375] Non-irradiated cells with nucleobase-photocaged RNA (3p-ds RNA + N3.1- basecage, -hv; GI2 II + N3.1 ,N3.2-basecage, -hv; GI2 II + N3.2,N3.5-basecage, -hv; and GI2 II + N3.1 ,N3.2,N3.5-basecage, -hv), respectively, showed significantly reduced levels of IFN-a production (dark grey bars, Figures 9B-9E) similar or basically identical to that observed for the negative control (neg Ctrl; black triangles, Figures OBOE). A more pronounced effect could be observed for cells transfected with nucleobase-photocaged RNA comprising two (GI2 II + N3.1 ,N3.2-basecage, +hv, (light grey bar, Figure 9C; GI2 II + N3.2,N3.5-basecage, +hv, light grey bar, Figure 9D) or more (GI2 II + N3.1 ,N3.2,N3.5-basecage, +hv, light grey bar, Figure 9E) nucleobase-photocages, whose level of IFN-a production almost matched that of the negative control RNA (neg Ctrl; black triangles, Figures 9B-9E).

[0376] This demonstrates that the nucleobase-photocaged dsRNA acts as RIG-I ligand after activation with light and that the photocage of the nucleobase at position N3.1 effectively inactivates (masks) essential molecular motifs needed for RIG-I activation, such as, e.g., RIG-l-mediated IFN responses. This masking effect is significantly enhanced by coupling of more than one nucleobase photocage to the ligand.

[0377] All in all, the results presented in this figure provide general proof of concept that photocaging of a nucleobase masks biological activity of the double-strandedoligonucleotide, and that the activity of the double-stranded oligonucleotide can be restored on demand in a spatiotemporally controlled manner by irradiation with light. Thus, the results presented in Figure 9B-9E provide general proof of concept that a conjugate according to the invention enables light-controlled targeted activation of RIG-I.Example 5:

[0378] By the results shown in Figure 10, the inventors further demonstrate that photocontrollable activation of RIG-I as indicated by ISRE reporter activation is also possible with conjugates according to the invention, which have a complementary RIG- I ligand (CRL) hybridized to a mRNA, in which the CRL comprises two photocages coupled to the nucleotides at the position N3.6 and N3.7 in the second strand (antisense strand) of the double-stranded oligonucleotide.

[0379] THP1 Dual™ wt cells (InvivoGen) were transfected with 800 ng / mL mRNA with or without prior hybridisation to a photo-controllable (“photocage-CRL”) or positive control (“CRL”) CRL using LipofectamineTM MessengerMaxTM (Invitrogen). Concentration was calculated on the mRNA amount. Samples were irradiated for 7 minutes with lightemitting diodes (365 nm, 2mW; grey) or not irradiated (0 min, black). 18 hours after transfection, supernatants were collected to determine the levels of Lucia luciferase induction by the interferone-stimulated response element (ISRE) reporter, which surrogates for the level of IFN production, stimulated by exposure to the sample RNAs using the Luciferase assay. The results are shown in Figure 10; they represent at least three experiments and mean ± SEM is shown.

[0380] “Cells only” and “mRNA only” negative controls showed no significant increase in immune stimulation, while, as expected, mRNA + CRL as positive control showed increased IFN induction regardless of light irradiation. The mRNA construct including the photocaged CRL showed an increase in IFN stimulation, comparable to the positive control only upon light irradiation. Without light irradiation the IFN induction of this construct remained as low as the negative controls.

[0381] All in all, the results presented in this figure provide general proof of concept that photocaging of a CRL hybridized to a mRNA masks the ability of IFN induction of the conjugate, and that the ability of IFN induction of the conjugate can be restored on demand in a spatiotemporally controlled manner by irradiation with light. Thus, the results presented in Figure 10 provide general proof of concept that a conjugate according to the invention enables light-controlled targeted activation of RIG-I and can be used as a light-inducible mRNA adjuvant.Example 6:

[0382] By the results shown in Figure 11 , the inventors further demonstrate that photocontrollable activation of RIG-I as indicated by IFN-expression levels is independent of the concentration of the conjugate used for transfection. This concentration independency is found for conjugates according of this invention, regardless of the number and positions of the photocages.

[0383] For the conjugates with one or more 2'-O-position coupled photocage(s) (A-B) peripheral blood mononuclear cells (PBMCs) were transfected with negative control RNA (neg Ctrl), RIG-I ligand control RNA (3p-dsRNA) or the following conjugate sample RNAs using Lipofectamine™2000 (Invitrogen): 3p-dsRNA + N3.7 2’0-photocage or 3p-dsRNA + N3.6+N3.7 2’0-photocage. These conjugate sample RNAs have a 2'-O- photocage in the second strand (antisense strand “N3”) at the following position(s), counted from the 5'-end of the first strand (i.e. 3' to 5' prime direction of the second strand): nucleotide 7 (N3.7), and nucleotides 6 and 7 (N3.6 + N3.7). On the x-axis indicated concentrations of each sample RNA were used for transfection. A part of the t...

Claims

CLAIMS1. A conjugate comprising:(a) at least one double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of at least eight nucleotides; and(ii) a second strand of a ribonucleic acid having a length of at least eight nucleotides and forming complementary basepairs with the first strand; wherein said at least one double-stranded oligonucleotide comprises at least one diphosphate or triphosphate at the 5'-end of the first strand or the second strand; and(b) at least one photoremovable protecting group (PPG); wherein the at least one PPG is coupled to a ribose at the 2'-O-position or a nucleobase of a nucleotide in the first or second strand.

2. The conjugate according to claim 1 , wherein the conjugate comprises two or more PPGs, each of which being independently coupled to a ribose at the 2'-O-position and / or nucleobase of a nucleotide in the first and / or second strand.

3. The conjugate according to claim 2, wherein:(i) at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the first strand and at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the second strand; or(ii) at least one PPG is coupled to a ribose at the 2'-O-position of a nucleotide in the first or second strand and at least one PPG is coupled to a nucleobase of a nucleotide in the first or second strand.

4. The conjugate according to claim 2, wherein:(i) each of the two or more PPGs is independently coupled to a ribose at the 2'-O- position of a nucleotide in the first strand; or(ii) each of the two or more PPGs is independently coupled to a ribose at the 2'-O- position of a nucleotide in the second strand.

5. The conjugate according to claim 2, wherein:(i) at least one PPG is coupled to a nucleobase of a nucleotide in the first strand and at least one PPG is coupled to a nucleobase of a nucleotide in the second strand; or(ii) each of the two or more PPGs is independently coupled to a nucleobase in the first strand; or(iii) each of the two or more PPGs is independently coupled to a nucleobase in the second strand.

6. The conjugate according to any one of the preceding claims, wherein the at least one double-stranded oligonucleotide comprises at least one diphosphate or triphosphate at the 5'-end of the first strand, such as a triphosphate at the 5'-end of the first strand.

7. The conjugate according to any one of the preceding claims, wherein the first and second strand of the at least one double-stranded oligonucleotide form a blunt-end at the 5 '-end of the first strand.

8. The conjugate according to any one of the preceding claims, wherein the first strand and / or the second strand of the at least one double-stranded oligonucleotide comprises at least one modification(s); preferably wherein the at least one modification(s) of the first strand and / or the second strand of the at least one double-stranded oligonucleotide is / are selected from the group consisting of deoxy- modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl- modification(s) or 2'-O-methoxyethyl-modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group-modification(s), locked nucleic acid (LNA)-modification(s), phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro- diamidate-morpholino-modification(s).

9. The conjugate according to any one of the preceding claims, wherein the PPG(s) may be the same or different at each occurrence.

10. The conjugate according to any one of the preceding claims, wherein the PPG(s) is / are coupled to the 2'-O-position of a ribose or a nucleobase, either directly by a photocleavable bond or via a photolabile self-immolative linker; preferably wherein the PPG(s) is / are coupled to (i) a ribose 2'-O-position via a photolabile self-immolative linker; or (ii) a nucleobase, either directly by a photocleavable bond or via a photolabile self-immolative linker.

11. The conjugate according to any one of the preceding claims, wherein the PPG is at each occurrence independently selected from the group of ortho-nitrobenzyl- based groups, ortho-nitro-2-phenethyl-based groups, (coumarin-4-yl)methyl-based groups, atto390-derived-coumarine-groups, benzocoumarin-based groups, 2- thionated coumarin-based groups, 2-dicyanomethylene coumarin-based groups, COUPY-based groups, cyanine(Cy)-based groups, porphyrin-based metal containing groups, boron dipyrromethen (BODIPY)-based groups, xanthene-based groups, pyronin-based groups, arylmethyl and arylcarbonylmethyl-based groups, 1 ,4-benzoquinone-based groups, bimane-based groups and 10H-phenothiazine- based groups; preferably wherein the PPG is at each occurrence independently selected from the group comprising the following groups (a1) to (a36):lerein is H, OMe or NMe2;lerein is S or C(=O);lereinRi is H and R2 is Br; orR1 is CH3 and R2 is H;lereindenotes the point of attachment of R;lereinRi is H and R2 is H;R1 is H and R2 is CH3;R1 is CH3 and R2 is H; orR1 is CH3 and R2 is CH3;lereindenotes the point of attachment of R;(a14)lereinR1 is CH, R2 is CH3 and X' is TfRi is N, R2 is CH3 and X' is Tf orR1 is CH, R2 is CeHi3 and X is Br;lereinR1 is OCH3, R2 is H and R3 is O;R1 is OCH3, R2 is H and R3 is NH;R1 is OCH3, R2 is H and R3 is S;R1 is OCH3, R2 is H and R3 is NOH;R1 is OCH3, R2 is CN and R3 is O;R1 is OCH3, R2 is CN and R3 is S;R1 is NEt2, R2 is H and R3 is O;R1 is NEt2, R2 is H and R3 is S;R1 is NEt2, R2 is CN and R3 is O; orR1 is NEt2, R2 is H and R3 is C(CN)2;lereinRi is CH3 and R2 is Et;R1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3 and R2 is Cl; orRi is CH3and R2 is I;(a20)lereinR1 is H and R2 is H;R1 is CH3 and R2 is H;R1 is CH3and R2 is CH3;orR1 is tBu and R2 is H;(a21)lerein is Zn, Pd, Cu or Ni;(a25)(a26)(a27)wherein(a) R = H and R1= NH2, NHCH3, NHEt, N(CH3)2or N(Et)2; or(b) R = Ome, NH2, NHCH3, NHEt, N(CH3)2or N(Et)2and R1= H;0(a28)(a29)wherein# denotes the point of attachment of the PPG to a photolabile self-immolative linker, a ribose 2'-O-position, a nucleobase N-atom or a nucleobase O-atom.

12. The conjugate according to any one of the preceding claims, wherein a nucleotide having a nucleobase with a coupled PPG comprises a structure selected from thewherein represents the coupled PPG and Rib represents the ribose moiety of the nucleotide.

13. The conjugate according to any one of claims 10 to 12, wherein the PPG coupled via a photolabile self-immolative linker comprises a structure selected from the group of:wherein X represents the point of attachment of the linker to the 2'-O-position or the nucleobase, and RL1, RL2, RL3and RL4are as defined in any one of the following (ia) to (id):(ia) RL1is H, RL2is H, RL3is CH3and RL4is H;(ib) RL1is H, RL2is Br, RL3is CH3and RL4is Br;(ic) RL1is H, RL2is H, RL3is NO2 and RL4is H; or(id) RL1is CH3O, RL2is H, RL3is CH3O and RL4is CH3O.

14. The conjugate according to any one of the preceding claims, wherein a nucleotide having a nucleobase with a coupled PPG comprises the structure:wherein a nucleotide with a PPG coupled to a ribose at the 2'-O-position comprises a structure selected from:wherein NB represents the nucleobase moiety of the nucleotide, such as15. The conjugate according to any one of the preceding claims, wherein the doublestranded oligonucleotide has a length of from 8 to 50 ribonucleotides, preferably a length of from 9 to 40 ribonucleotides, more preferably of from 11 to 30 ribonucleotides, even more preferably of 11 ribonucleotides or 20 to 24 ribonucleotides.

16. The conjugate according to claim 15, wherein(i) the double-stranded oligonucleotide is blunt-ended; and / or(ii) the first strand or the second strand of the double-stranded oligonucleotide comprises at least one modification(s); preferably wherein the at least one modification(s) of the first strand or the second strand of the double-stranded oligonucleotide is / are selected from the group consisting of deoxy-modification(s), O-methyl-group-modification(s), preferably 2'-O-methyl-modification(s) or 2'-O-methoxyethyl- modification(s), fluoro-group-modification(s), preferably 2'-fluoro-group- modification(s), locked nucleic acid (LNA)-modification(s),phosphodiester-modification(s), peptide nucleic acid modification(s) and phosphoro-diamidate-morpholino-modification(s); and / or(iii) the at least one PPG(s) may be the same or different at each occurrence.

17. The conjugate according to claim 16, wherein at least one PPG(s) is / are coupled to the 2'-O-position by a photolabile self-immolative linker; preferably(A) wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1, position 2, position 4, position 5, position 6, position 7, position 8, position 9, position 14, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, position 11, position 12, position 13, position 24, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii); or(B) wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1 , position 5, position 6, position 7, position 8, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and (ii); or(C) wherein one PPG is coupled at position 1 of the first strand, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of the first strand selected from position 5, position 6, position7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 6, position 7, position 8, and any combination thereof; or(D) wherein one PPG is coupled at position 6 (in a 3' to 5' prime direction), one PPG is coupled at position 7 (in a 3' to 5' prime direction) of the second strand, and at least one PPG(s) is / are, optionally, coupled at:(i) a position of the first strand selected from position 1 , position 5, position6, position 7, position 8, and any combination thereof; and / or(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 2, position 5, position 8, and any combination thereof; preferablywherein one PPG is coupled at position 6 (in a 3' to 5' prime direction) and one PPG is coupled at position 7 (in a 3' to 5' prime direction) of the second strand.

18. The conjugate according to any one of claims 15 to 17, wherein at least one PPG(s) is / are coupled to a nucleobase by a photocleavable bond or via a photolabile self- immolative linker, preferably by a photocleavable bond; preferably wherein the at least one PPG(s) is / are coupled at:(i) a position of the first strand selected from position 1 , position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof;(ii) a position of the second strand, in a 3' to 5' prime direction, selected from position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and any combination thereof; or(iii) two or more PPGs are coupled to the first and / or second strand at positions selected from (i) and / or (ii); preferably wherein the conjugate comprises 2, 3, 4 or 5 PPGs coupled to a nucleobase in the first and / or second strand; preferably wherein nucleotides having a PPG coupled to a nucleobase are spaced apart from one another by 1, 2 or 3, preferably 1 or 2, nucleotides that are free from a PPG coupled to a nucleobase; and / or wherein at least one PPG(s) is / are coupled at position 1 of the second strand and / or at position 2 of the second strand and / or at position 5 of the second strand, such as at positions 2 and 5 or positions 1 , 2 and 5 of the second strand, or at positions 3 and 5 of the second strand.

19. The conjugate according to claim 15 and claim 16(H) to 17, wherein the doublestranded oligonucleotide has a length of from 8 to 17 ribonucleotides, at least one diphosphate or triphosphate, preferably a triphosphate, at the 5'-end of at least the first or second strand, and further comprises a single-stranded overhang at at least one 3'-end of at least the first or second strand and / or at the 5'-end of the strand which does not have a di- / triphosphate, wherein the single-stranded overhang comprises at least one guanosine.

20. The conjugate according to claim 19, wherein the double-stranded oligonucleotide has a length of from 8 to 15, 8 to 12, 10 to 12, or 11 ribonucleotides, preferably 10 to 12, or 11 ribonucleotides; and / orwherein the at least one di- / triphosphate is at the 5'-end of the strand of the double-stranded oligonucleotide which also comprises the single-stranded overhang; and / or wherein the single-stranded overhang has a length of at least 3 nucleotides; preferably wherein the single-stranded overhang has a length of 3 to 15 nucleotides, 3 to 10 nucleotides, 3 to 8 nucleotides or 3 to 5 nucleotides; and / or wherein the single-stranded overhang comprises at least two guanosines or at least three guanosines; and / or wherein the single-stranded overhang has a length of 3 to 5 nucleotides and comprises at least three guanosines, such as three consecutive guanosines (GGG); and / or wherein the single-stranded overhang comprises or consists of 5 nucleotides having the sequence adenosine-adenosine-guanosine-guanosine-guanosine (AAGGG); and / or wherein the first and second strand of the double-stranded oligonucleotide form at least a blunt-end at the 5'-end of the first strand; and / or wherein the double-stranded oligonucleotide comprises a further overhang, preferably a further overhang at the strand not comprising the single-stranded overhang comprising at least one guanosine; preferably wherein the further overhang is a single-stranded RNA-overhang or an overhang with a fluorophore or a lipophilic substitution.

21. The conjugate according to claim 19 or 20, wherein the at least one doublestranded oligonucleotide is connected with the single-stranded overhang via a linker moiety L; preferably wherein the linker moiety L is a nucleotide-linker or a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms; such as wherein the linker moiety L is a nucleotide-linker; preferably a nucleotide-linker having a length of at least 2 nucleotides; more preferably a nucleotide-linker having a length of 2 to 10 nucleotides; even more preferably wherein the nucleotide-linker is an adenosine- linker, preferably with at least 2 adenosines, more preferably with 2 adenosines; or a hydrocarbon-based linker according to: (i) Formula IFormula I wherein n is an integer in the range of from 3 to 8, preferably in the range of from5 to 7; or (ii) Formula IIFormula II wherein n is an integer in the range of from 3 to 8, preferably in the range of from 5 to 7; more preferably wherein n is 6.

22. The conjugate according to any one of claims 1 to 14, wherein the at least one double-stranded oligonucleotide comprised in the conjugate comprises a structure according to Formula III, Formula IV or Formula V:RNA1 - BL1 - RNA3 RNA1 RNA3RNA2 RNA4 RNA2 - BL2 -RNA4Formula III Formula IVRNA1 - BL1 - RNA3RNA2 - BL2 - RNA4Formula V whereinRNA1 represents the first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA2 represents the second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the first strand via complementary base pairing;RNA3 represents a third strand of a ribonucleic acid having a length of from 8 to 50 nucleotides;RNA4 represents a fourth strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forms a double-stranded oligonucleotide with the third strand via complementary base pairing;BL1 , if present, represents a bivalent linker that covalently bonds the 3'-end of RNA1 to the 3'- or 5'-end of RNA3;BL2, if present, represents a bivalent linker that covalently bonds the 5 -end of RNA2 to the respective 5'- or 3'-end of RNA4; whereinRNA1 and RNA2, as well as RNA3 and RNA4, each have no overhang of the 5’- terminal nucleotide residues, and wherein RNA1 and RNA2, as well as RNA3 and RNA4, each can comprise an overhang of the 3’-terminal nucleotide residues of not more than five nucleotides, preferably of not more than four or three nucleotides, more preferably of not more than two nucleotides, even more preferably of not more than one nucleotide, especially no overhang of the 3’-terminal nucleotide residues.

23. The conjugate according to claim 22, wherein the linker BL1 and BL2, each independent from each other, is selected from the group of a nucleotide-linker as defined in claim 21 , a hydrocarbon-based linker, preferably comprising 3 to 24 main chain carbon atoms, preferably comprising 6 to 18 main chain carbon atoms as defined in claim 21 , a phosphodiester linker (OPO(OH)O-), and any combination thereof; and / or wherein the sequence of RNA1 is identical to the sequence of RNA3; and / or wherein the sequence of RNA2 is identical to the sequence of RNA4; and / or wherein each of RNA1 , RNA2, RNA3 and RNA4 has: (i) a length of from 9 to 40 nucleotides, preferably of from 11 to 30 nucleotides, more preferably of from 20 to 24 nucleotides; or (ii) a length of from 8 to 11 nucleotides, preferably 11 nucleotides; and / or wherein the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 may have the same or a different length; and / or wherein the dimer unit RNA1 / RNA2 and the dimer unit RNA3 / RNA4 each are blunt-ended double-stranded RNA.

24. The conjugate according to any one of claims 1 to 14, wherein the conjugate comprises a structure according to Formula VI:Formula VI wherein sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of a mRNA strand;L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms; and dsO represents a double-stranded oligonucleotide, comprising: (i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and (ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand; or wherein the conjugate comprises a structure according to Formula VII:Formula VII wherein mRNA is a mRNA-strand;(sN-L3-dsO) represents a unit comprising (a) to (c), wherein(a) sN represents a single-stranded nucleic acid sequence capable of hybridizing to at least one nucleic acid sequence of the mRNA-strand;(b) L3, if present, represents a linker comprising 3 to 24, preferably 6 to 18, main chain atoms;(c) dsO represents a double-stranded oligonucleotide, comprising:(i) a first strand of a ribonucleic acid having a length of from 8 to 50 nucleotides; and(ii) a second strand of a ribonucleic acid having a length of from 8 to 50 nucleotides and forming complementary base pairs with the first strand; and m is an integer in the range of from 1 to 4; preferably wherein m is 2, 3 or 4, preferably m is 4.

25. The conjugate according to any one of claims 1 to 24 for use as a medicament.

26. The conjugate according to any one of claims 1 to 24, such as the conjugate comprising a structure according to Formula VII, for use as a vaccine.

27. Pharmaceutical composition comprising the conjugate according to any one claims 1 to 24 and, optionally, at least one pharmaceutically acceptable carrier, excipient or delivery agent.

28. Drug delivery system comprising the conjugate according to any one of claims 1 to 24, characterized in that the conjugate is a caged drug comprising the doublestranded oligonucleotide as therapeutic agent that is rendered biologically inactive by being coupled to at least one PPG(s); wherein said double-stranded oligonucleotide is capable of being activated and / or released in response to a predetermined wavelength and / or intensity of light which removes the at least one coupled PPG(s) tracelessly.

29. The conjugate according to any one of claims 1 to 24 for use as a medicament.

30. The conjugate according to any one of claims 1 to 24, the pharmaceutical composition of claim 28 or the drug delivery system according to claim 29 for use in a method of treatment or prevention of a disease, comprising: administering the conjugate or system to a patient, localising the conjugate or system at the target site, and directing a predetermined wavelength and / or intensity of light to the target site to activate and / or release the caged double-stranded oligonucleotide as therapeutic agent at the target site.

31. The conjugate, composition or system for use according to claim 30, wherein the conjugate, composition or system is administered (i) orally or (ii) parenterally, such as by intravenous injection, intratumoral injection, intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, sublingual application, buccal application, intranasal application, rectal application, vaginal application, ocular route, otic route, inhalation through the nose, mouth, or a combination thereof; application to the skin, transdermal application, or any combination of the foregoing; to the patient.

32. The conjugate, composition or system for use according to any one of claims 29 to 31 , wherein the disease is cancer or an infection, preferably a virus infection.

33. The conjugate, composition or system for use according to any one of claims 29 to 32, 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 tumor (PNET)), and combinations thereof.

34. The conjugate, composition or system for use according to any one of claims 29 to 32, wherein the disease is a hematological tumor or a solid tumor.

35. The conjugate according to any one of claims 1 to 24 for use in a method of infiltrating immune cells, preferably T cells.

36. The conjugate according to any one of claims 1 to 24 for use in a method of turning cold into hot tumors.

37. Use of the conjugate according to any one of claims 1 to 24 in the manufacture of a medicament for the treatment or prevention of a disease, such as a disease as defined in any one of claims 32 to 34.

38. A method of treating a disease, comprising the step of administering a therapeutically effective amount of the conjugate according to any one of claims 1 to 24, the pharmaceutical composition of claim 28 or the drug delivery system according to claim 29 to a subject in need thereof, and directing a predetermined wavelength and / or intensity of light to a target site to activate and / or release the caged doublestranded oligonucleotide as therapeutic agent at the target site.

39. The method of claim 38, wherein the disease is a disease as defined in any one of claims 32 to 34.

40. A kit for use in medicine comprising the conjugate according to any one of claims 1 to 24; such as 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 as defined in any one of claims 32 to 34.

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