Boosting cap-independent translation

Abstract

The present invention relates to an enhancer of IRES mediated translation, wherein the enhancer is an inhibitor of cap-dependent translation.

Description

[0001] Boosting cap-independent translation

[0002] The present invention relates to an enhancer of IRES mediated translation, wherein the enhancer is an inhibitor of cap-dependent translation.

[0003] Background of the Invention

[0004] Translation initiation is a rate-limiting step of protein synthesis. It is highly regulated by different mechanisms, depending on the structural distinction of mRNAs. Most cellular mRNAs are translated by a cap-dependent mechanism that requires the binding of the trimeric complex eukaryotic initiation factors (eIF)4F, comprised of eIF4G, eIF4E, and eIF4A, to the 5’ 7-methyl GpppN (cap) structure at the 5' end of the mRNA.

[0005] De novo initiation of typical mRNA requires recognition of the cap structure by the trimeric translation factor complex eIF4F, and subsequent recruitment of a 43 S ribosomal subunit (containing a 40S ribosomal subunit, eukaryotic initiation factors (elFs) elFl, elFla, eIF2, eIF5, eIF3 and Met-tRNAiMet) and other initiation factors to form a 48S ribosomal preinitiation complex. The 48S complex is functional for scanning the mRNA sequence in a 5 '-3' direction for initiation codons in a favorable consensus sequence. The initiation phase of translation is completed when the 60S ribosomal subunit has joined, and then the 80S ribosome completes the translation of the mRNA. eIF4F is a heterotrimeric complex consisting of eIF4G, eIF4E and eIF4A, and the primary function of eIF4F is to facilitate binding of 40S ribosomal subunits to the 5' cap structure of mRNA and then aid ribosomal scanning. Because eIF4G can simultaneously bind all these initiation factors, it performs two critical linking or bridging functions. First, eIF4G- mediated linkage of eIF3 (which is bound to 40S ribosomal subunits), and eIF4E, completes a molecular bridge which binds the mRNA to the ribosome. Second, eIF4G-mediated linkage of PABP and eIF4E simultaneously provides a second molecular bridge linking 5' and 3' ends of the mRNA in a pseudo-circularized structure (Wells et al. Mol Cell. Jul 1998;2(1): 135-40). Thus, eIF4G is in many ways the centerpiece of the translation initiation complex. eIF4E is the least abundant of all initiation factors and is a major target for translational control. Mammalian eIF4E activity is modulated by its reversible association with a family of three related polypeptides termed 4E-binding proteins (4EBP). The eIF4E / 4EBPs interaction is regulated by phosphorylation of the 4EBPs. Hypo-phosphorylated 4EBPs bind tightly to eIF4E, resulting in an inhibition of cap-dependent translation. The 4EBPs do not inhibit eIF4E binding to the cap, but instead block eIF4F assembly by competing with eIF4Gs for a common binding site on eIF4E. Thus, 4EBPs act as molecular mimics of eIF4G.

[0006] Some viral mRNAs have evolved cap-independent mechanisms of translation initiation including the use of internal ribosome-entry sites (IRES) sequences, ribosome shunting or VpG shunting. By virtue of being cap-independent, this type of translation depends on a different set of initiation factors, and is generally independent of eIF4E. IRES mediated translation is also of relevance for transcripts devoid of a 5’ cap such as circular RNAs (circRNA).

[0007] Circular RNA are long non-coding RNAs characterized as covalently closed molecules. CircRNAs are typically produced by a non-linear ‘backsplicing’ event using a downstream splice donor (SD) and an upstream splice acceptor (SA) in contrast to conventional linear splicing. It is widely appreciated that circRNAs by virtue of their circular nature are resistant towards exonucleolytic decay, and therefore circRNAs comprise a very stable class of RNA with half-lives greatly exceeding that of conventional linear mRNA and consequently, the therapeutic potential of a durable circular RNA concept with engineered functionalities has now emerged. While circRNAs are devoid of 5’cap and 3’ polyA tails and thus per se are not substrates for translation, the insertion of IRES sequences effectively converts non-coding circRNAs into highly effective protein-encoding molecules.

[0008] Thus IRES mediated translation is of importance for transcripts such as circRNAs that are not subject to cap-dependent translation. The present invention provides an enhancer of IRES-mediated translation that depends on the inhibition of cap-dependent translation.

[0009] Summary of the Invention

[0010] In a first aspect, the present invention provides an enhancer of IRES mediated translation, wherein the enhancer is an inhibitor of cap-dependent translation.

[0011] In a second aspect, the present invention provides a method for increasing translation of a circular RNA comprising the steps of

[0012] (a) providing an enhancer of IRES-mediated translation according to the first aspect of the invention; and

[0013] (b) administering said enhancer to cells comprising an IRES operably linked to the circular RNA to increase translation of the circular RNA.

[0014] In a third aspect, the present invention provides a nucleic acid encoding the enhancer of IRES-mediated translation according to the first aspect of the invention and optionally further encoding a circular RNA operably linked to an IRES. In a fourth aspect, the present invention provides a vector encoding a nucleic acid according to the third aspect of the invention.

[0015] List of Figures

[0016] In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and / or below.

[0017] Figure 1: refers to (A) Western blot analysis of ICOSL protein expression from A375 cells transfected with circRNA (circ)ICOSL expression plasmids and treated with 4EGI-1 at the indicated concentration. Beta-actin was used as loading control. (B) RT-qPCR of circICOSL expression from A375 cells transfected with circICOSL expression plasmids and treated with 4EGI-1 at the indicated concentration, relative to GAPDH mRNA. (C) Western blot analysis of circRNA (circ)eGFP protein expression from A375 cells transfected with circEGFP expression plasmids and treated with 4EGI-1 at the indicated concentration. Betaactin was used as loading control. Graph depicts eGFP protein expression relative to Beta-actin. (D) RT-qPCR of circEGFP expression from A375 cells transfected with circEGFP expression plasmids and treated with 4EGI-1 at the indicated concentration, relative to GAPDH mRNA.

[0018] Figure 2: refers to (A) Western blot analysis of eGFP protein expression from A375 cells transfected with circEGFP expression plasmids and N FLAG 4EBP1 phospho-mutant plasmids or empty vector control. Beta-actin was used as loading control. (B) RT-qPCR of circEGFP expression from A375 cells transfected with circEGFP expression plasmids and N FLAG 4EBP1 phospho-mutant plasmids or empty vector control, relative to GAPDH mRNA. (C) Western blot analysis of eGFP protein expression from A375 cells transfected with circEGFP expression plasmids and mCherry_IRES_N_FLAG_4EBPl phospho-mutant plasmids or empty vector control. Beta-actin was used as loading control. (D) RT-qPCR analysis of circEGFP expression from A375 cells transfected with circEGFP expression plasmids and and mCherry_IRES_N_FLAG_4EBPl phos-mutant plasmids or empty vector control., relative to GAPDH mRNA.

[0019] Figure 3: refers to (A) Western blot analysis of eGFP protein expression from A375 cells co-transfected with circEGFP plasmids and siRNA targeting eIF4E (eIF4E_KDl = SEQ ID NO: 0018); eIF4E_KD2 = SEQ ID NO: 0019) or control siRNA. Beta-actin used as loading control (Left). Graph depicting eGFP protein expression of eGFP relative to Beta-actin loading control. Data for 3 independent experiments shown (Right). (B) RNA levels of circEGFP and eIF4E assessed by RT-qPCR relative to GAPDH mRNA, n=3. Data for 3 independent experiments shown. Figure 4: refers to (A) Western blot analysis of eGFP and Renilla protein expression from A375 cells co-transfected with circEGFP and linear Renilla mRNA expression plasmids and N FLAG IRES booster experiments, as denoted, or empty vector control. Beta-actin used as loading control (Left). Graph depicting eGFP protein expression of eGFP relative to Betaactin loading control (Right). (B) RNA levels of circEGFP and Renilla mRNA assessed by RT-qPCR relative to GAPDH mRNA. (C) Western blot analysis of eGFP and Renilla protein expression from A375 cells co-transfected with circEGFP and linear Renilla mRNA expression plasmids and N FLAG IRES booster experiments, as denoted, or empty vector control. Betaactin used as loading control (Left). Graph depicting eGFP protein expression of eGFP relative to Beta-actin loading control (Right). (D) RNA levels of circEGFP and Renilla mRNA assessed by RT-qPCR relative to GAPDH mRNA.

[0020] Figure 5: refers to Figure 5: (A) Western blot analysis of eGFP and Firefly protein expression from A375 cells co-transfected with circEGFP and linear Firefly mRNA expression plasmids and N FLAG IRES booster experiments, as denoted, or empty vector control. Betaactin used as loading control (Left). Graph depicting eGFP protein expression of eGFP relative to Beta-actin loading control (Right). (B) RNA levels of circEGFP and Firefly mRNA assessed by RT-qPCR relative to GAPDH mRNA.

[0021] Detailed Descriptions of the Invention

[0022] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0023] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0024] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

[0025] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference ” . In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0026] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0027] Definitions

[0028] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0029] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

[0030] The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

[0031] The term “circular RNA” or “circRNA”, as used herein, refers to a type of RNA in which the ends of the RNA strand have been covalently joined to form a closed continuous loop. CircRNAs are generally formed by covalent binding of the 5’ site of an upstream exon with the 3’ site of the same or a downstream exon.

[0032] The term “Internal ribosomal entry site (IRES)”, as used herein, refers to regions in the RNAs that allow the internal initiation of translation in a cap-independent manner. An IRES element typically comprises a stretch of highly structured RNA containing several stem-loop structures. IRESs have initially been identified in picornavirus, but they are present in a variety of different virus. More recently IRES sequences have also been identified in many cellular mRNAs. Both types of IRES sequences (i.e. viral and cellular IRES sequences) generally can be used to practice the present invention.

[0033] Viral IRESs can be divided into four distinct classes based on two major criteria: first, the type of secondary and tertiary structures of their RNA elements and second, their mode of action for translation initiation (see Mailliot and Martin, RNA 2017, el 458).

[0034] Class 1 IRESs usually require all initiation factors except eIF4E and contain rather basic secondary structures consisting of short and long hairpins. Class 1 IRESs typically recruit the ribosome upstream of the coding region and rely on a classical 5’ to 3’ scanning to find the start codon. Class 1 IRESs are found for example in: Enterovirus A71 (EV-A71), Coxsackievirus B3 (CVB3), poliovirus (PV), and Human Rhino Virus 2 (HRV).

[0035] In contrast, class 2 IRESs promote direct tethering of the translation initiation machinery directly to the start codon and without any scanning step. Class 2 IRESs are found for example in: Picornaviridae such as encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV).

[0036] Class 3 IRESs contain more sophisticated secondary and tertiary structures such as pseudoknots. They require only a small subset of translation initiation factors, namely eIF2, eIF3, and eIF5 to recruit the ribosome and load it directly on the AUG start codon without scanning. Class 3 IRESs are found for example in: Flaviviridae family such as hepatitis C virus (HCV) and classical swine fever virus (CSFV); Picornaviridae, such as porcine teschovirus and porcine enterovirus 8 (PEV8) or simian virus 2 (SV2).

[0037] Class 4 IRESs are the more compact and sophisticated IRESs in term of structural complexity; they contain usually several pseudoknots and do not require any translation initiation factors. They are the smallest IRESs known (usually less than 200 nucleotides). Class 4 IRESs are found for example in: Dicistroviridae such as cricket paralysis virus (CrPV), Israeli acute paralysis virus (IAPV), Platia Stali instestine virus (PSIV), or Taura syndrome virus (TSV). The most preferred class of IRESs in the context of the present invention are class 1 and class 2 IRESs.

[0038] The term “cap-dependent translation initiation” as used herein, refers to the first step of cap-dependent protein translation. In general, cap-dependent protein translation can be divided into three main steps: initiation, extension, and termination. Initiation is the rate limiting step in cap-dependent translation, and the formation of the translation initiation complex (eIF4F) is the most critical process at this step.

[0039] The terms “translation initiation complex” or “eIF4F” as used herein refers to a heterotrimeric complex of eIF4A, eIF4E, and eIF4G. eIF4A is a helicase that expands RNA into a single strand through the RNA binding protein eIF4B. eIF4E is a cap-binding protein and eIF4G is a scaffolding protein used for mechanical assembly. eIF4F assembles on the structure of 5' capped mRNA and interacts with polyA tail-binding protein (PABP) and is thus important for recruiting the 40S ribosomal subunit to the 5 ’cap of mRNA.

[0040] The term “decapping enzyme” as used herein refers to enzymes that are capable of removing the 5’cap structure of mRNAs (i.e. ‘decapping’). Typically, decapping enzymes include nudix hydrolase motifs that are necessary for liberating m7GDP from capped mRNAs. Preferred decapping enzymes are D9 and DIO from Vaccinia virus.

[0041] The term “4EBP” or “4E-binding protein” as used herein refers to a family of proteins that interact with eIF4E (which is part of the translation initiation complex). The 4EBPs are regulated by phosphorylation, whereby dephosphorylated 4EBPs bind strongly to eIF4E and thereby inhibit formation of the translation initiation complex. Phosphorylated 4EBPs in contrast only binds weakly to eIF4E and does not inhibit cap-dependent translation. Preferred 4EBP proteins are 4EBP1, 4EBP2 and 4EBP3.

[0042] The term “small molecule drug” as used herein, refers to a low molecular weight (< 900 Daltons) compound.

[0043] The term “4EGI-1” refers to a small molecule inhibitor of eIF4E / eIF4G interaction, with a Kd of 25 pM against eIF4E binding. 4EGI-1 has the following chemical structure:

[0044] 4EGI-1 (CAS: 315706-13-9)

[0045] The term “4ElRCat” refers to a small molecule inhibitor of eIF4E / eIF4G interaction, with a IC50 of 4 pM. 4ElRCat has the following chemical structure:

[0046] The term “4E2RCat” refers to a small molecule inhibitor of eIF4E / eIF4G interaction, with a IC50 of 13.5 iM. 4ElRCat has the following chemical structure:

[0047] The term “allolaurinterol” refers to a small molecule inhibitor of eIF4A. Allolaurinterol has the following chemical structure: Allolaurinterol (CAS: 62311-74-4)

[0048] The term “Elisabatin A” refers to a small molecule inhibitor of eIF4A. Elisabatin A has the following chemical structure:

[0049] Elisabatin A (CAS: 237749-84-7)

[0050] The term “Rocaglamide A” refers to a small molecule inhibitor of eIF4A. Rocaglamide

[0051] A has the following chemical structure:

[0052] Rocaglamide A (CAS: 84573-16-0)

[0053] The term “Silvestrol” refers to a small molecule inhibitor of eIF4A. Silvestrol has the following chemical structure: Silvestrol (CAS: 697235-38-4)

[0054] The term “Pateamine” refers to a small molecule inhibitor of eIF4A. Pateamine has the following chemical structure:

[0055] (Pateamine (CAS: 139220-18-1)

[0056] The term “Hippuristanol” refers to a small molecule inhibitor of eIF4A. Hippuristanol has the following chemical structure: Hippuristanol (CAS: 80442-78-0)

[0057] The term “Ribavirin” refers to a small molecule inhibitor of eIF4E that competes with m7cap for eIF4E binding. Ribavirin has the following chemical structure:

[0058] Ribavirin (CAS: 36791-04-5)

[0059] Embodiments

[0060] In the following different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0061] In a first aspect, the present invention provides an enhancer of IRES mediated translation, wherein the enhancer is an inhibitor of cap-dependent translation.

[0062] In a preferred embodiment of the first aspect of the present invention, the enhancer is an inhibitor of cap-dependent translation initiation. In general, any inhibitor targeting the initiation of cap-dependent translation initiation could be used as an enhancer of IRES mediated translation.

[0063] In a preferred embodiment, the inhibitor of cap-dependent translation initiation is selected from the group comprising: an inhibitor of the eIF4F complex and a decapping enzyme.

[0064] In a preferred embodiment, the enhancer of IRES mediated translation is an inhibitor of the eIF4F complex (translation initiation complex). Inhibitors of the eIF4F complex could either target the complex as such (e.g. inhibiting assembly of the complex, inhibiting binding between compounds of the complex) and / or being directed to specific compounds of the complex (e.g. eIF4G, eIF4E or eIF4A). In a preferred embodiment, the enhancer is an inhibitor of eIF4G. In a preferred embodiment, the enhancer is an inhibitor of eIF4E. In a preferred embodiment, the enhancer is an inhibitor of eIF4A.

[0065] In a preferred embodiment, the enhancer of IRES mediated translation is a decapping enzyme.

[0066] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by interfering with

[0067] (a) interaction of eIF4E with eIF4G; and / or

[0068] (b) binding of eIF4E to capped mRNA.

[0069] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by inhibiting the interaction of eIF4G and polyA tail-binding protein (PABP).

[0070] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by interfering with interaction of eIF4A with eIF4G.

[0071] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by interfering with interaction of eIF4E with eIF4G.

[0072] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by interfering with binding of eIF4E to capped mRNA.

[0073] In a preferred embodiment, the enhancer of IRES-mediated translation inhibits the eIF4F complex by interfering with interaction of eIF4E with eIF4G and binding of eIF4E to capped mRNA.

[0074] In a preferred embodiment, the enhancer directly interacts with eIF4E.

[0075] In a preferred embodiment, the enhancer is selected from a small molecule, a protein and a nucleic acid. In general any structural type of compound can be used to exercise the present invention. In a preferred embodiment, the enhancer is a small molecule.

[0076] In a preferred embodiment, the enhancer is a protein. In a preferred embodiment, the protein is:

[0077] (i) competing with eIF4G for binding to eIF4E, preferably the protein is a 4E- binding protein (4EBP);

[0078] (ii) a decapping enzyme, preferably a decapping enzyme from Vaccinia Virus; or

[0079] (iii) decreasing phosphorylation of a 4E-binding protein (4EBP), preferably the protein is a SV40 small T-antigen.

[0080] In a preferred embodiment, the protein is competing with eIF4G for binding to eIF4E, preferably being a 4E-binding protein (4EBP). In a preferred embodiment, the protein is a decapping enzyme. In a preferred embodiment, the decapping enzyme is a decapping enzyme from Vaccinia Virus. In a preferred embodiment the decapping enzyme is selected from Vaccinia Virus D9 and DIO. In a preferred embodiment the decapping enzyme is Vaccinia Virus D9. In a preferred embodiment the decapping enzyme is Vaccinia Virus DIO. In a preferred embodiment the decapping enzyme comprises or consists of the amino acid sequence according to SEQ ID NO: 00012 and 00014. In a preferred embodiment the decapping enzyme is encoded by the sequences selected from SEQ ID NO: 00011 and SEQ ID NO: 00013.

[0081] In a preferred embodiment, the protein is decreasing phosphorylation of a 4E-binding protein (4EBP). In a preferred embodiment, the protein decreasing phosphorylation of a 4E- binding protein (4EBP) is SV40 small T-antigen. In a preferred embodiment, the SV40 small T-antigen comprises or consists of the amino acid according to SEQ ID NO: 008 or SEQ ID NO: 0010. In a preferred embodiment, the SV40 small T-antigen comprises or consists of the amino acid according to SEQ ID NO: 008. In a preferred embodiment, the SV40 small T- antigen comprises or consists of the amino acid according to SEQ ID NO: 0010. In a preferred embodiment the SV40 small T-antigen is encoded by the nucleic acid sequence according to SEQ ID NO: 007 or SEQ ID NO: 009.

[0082] In a preferred embodiment, the enhancer of IRES-mediated translation is a 4EBP1 mutant protein, wherein at least one phosphorylation site selected from Thr37, Thr46, Ser65, Thr70, Ser83, SerlOl, and Seri 12 of human 4EBP1 (Uniprot NO: Q13541) is inactivated. In a preferred embodiment, the 4EBP1 mutant protein comprises at least the inactivated phosphorylation sites Thr37 and Thr46 of human 4EBP1 (Uniprot NO: Q13541). Several phosphorylation sites within human 4EBP1 (UniprotNO: Q13541) have been identified: Thr37, Thr46, Ser65, Thr70, Ser83, SerlOl, and Seri 12. The first five phosphorylation sites are phylogenetically conserved among all species, and phosphorylation of Thr37 and Thr46 serves as a priming event, which is followed by Thr70 phosphorylation and finally Ser65 phosphorylation. In a preferred embodiment the 4EBP1 mutant protein comprises a sufficient number of inactivated phosphorylation sites (e.g. 1, 2, 3, 4, 5, 6, 7 or 8 inactivated phosphorylation sites) to prevent the priming event, thus avoiding inactivation of the 4EBP1 mutant protein by phosphorylation. In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46, Ser65, Thr70, Ser83, SerlOl, and Seri 12 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46, Ser65, Thr70, Ser83 and SerlOl of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46, Ser65, Thr70 and Ser83 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46, Ser65 and Thr70 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46 and Thr70 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37, Thr46 and Ser65 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation sites Thr37 and Thr46 of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises the following inactivated phosphorylation site Thr37 of human 4EBP1 (Uniprot NO: Q13541).

[0083] In a preferred embodiment, the enhancer of IRES-mediated translation is a 4EBP1 mutant protein, wherein the 4EBP1 mutant protein comprises at least substitutions T37A and T46A of human 4EBP1 (Uniprot NO: Q13541) and optionally further comprises S65A and T70A substitutions. A substitution indicated as for example T37A, indicated that on position 37 of the amino acid sequence a substitution replacing amino acid T (threonine) with amino acid A (alanine). In a preferred embodiment, the 4EBP1 mutant protein comprises at least substitutions T37A and T46A of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises at least substitutions T37A, T46A and S65A of human 4EBP1 (Uniprot NO: Q13541). In a preferred embodiment, the 4EBP1 mutant protein comprises at least substitutions T37A, T46A, S65A and T70A of human 4EBP1 (Uniprot NO: Q13541). The present invention is not limited to amino acid substitutions with amino acid A (alanine). The amino acid of the phosphorylation site could also be inactivated by substitutions with other amino acids. In a preferred embodiment, the phosphorylation site is inactivated by a conservative amino acid substitution. In a preferred embodiment, the amino acid of the phosphorylation site is substituted with a non-proteinogenic amino acid. In another embodiment, the phosphorylation site is inactivated by deletion of the amino acid.

[0084] In a preferred embodiment, the 4EBP1 mutant protein comprises the amino acid sequence according to SEQ ID NO: 004 or SEQ ID NO: 006. In a preferred embodiment, the 4EBP1 mutant protein comprises the amino acid sequence according to SEQ ID NO: 004. In a preferred embodiment, the 4EBP1 mutant protein comprises the amino acid sequence according to SEQ ID NO: 006. In a preferred embodiment, the 4EBP1 mutant protein is encoded by the nucleic acid sequence according to SEQ ID NO: 003 or SEQ ID NO: 005. In a preferred embodiment, the enhancer of IRES-mediated translation is a nucleic acid. Generally, the nucleic acid can either be an active entity itself (e.g. siRNA, shRNA or microRNA) or encode a protein which is the enhancer. In a preferred embodiment, the nucleic acid is a siRNA, shRNA or microRNA. In a preferred embodiment, the nucleic acid is a siRNA. In a preferred embodiment, the nucleic acid is a microRNA. In a preferred embodiment, the nucleic acid is a shRNA. In some embodiments, the nucleic acid encodes one of the proteins disclosed herein as enhancer of IRES-mediated translation.

[0085] In a preferred embodiment, the enhancer of IRES-mediated translation is siRNA, shRNA or microRNA (preferably siRNA) targeting at least one component of the eIF4F complex (i.e. eIF4G, eIF4E and eIF4A). In a preferred embodiment, the enhancer of IRES- mediated translation is siRNA directed to eIF4E. In a preferred embodiment, the nucleic acid has the sequence as specified in SEQ ID NO: 0018 or SEQ ID NO: 0019. In a preferred embodiment, the enhancer of IRES-mediated translation is siRNA directed to eIF4G. In a preferred embodiment, the enhancer of IRES-mediated translation is siRNA directed to eIF4A. In a preferred embodiment, the enhancer of IRES-mediated translation is a DNA expression vector encoding one of the proteins, or a trans-dominant mutant hereof, disclosed herein. In a preferred embodiment, the enhancer of IRES-mediated translation is a DNA expression vector encoding a decapping enzyme. In a preferred embodiment, the enhancer of IRES-mediated translation is a DNA expression vector encoding an inhibitor of cap-dependent translation. In a preferred embodiment, the DNA expression vector encoding a circular RNA operably linked to an IRES is also encoding the enhancer of IRES-mediated translation.

[0086] In a preferred embodiment, the enhancer of IRES-mediated translation is a small molecule. In a preferred embodiment, the enhancer of IRES-mediated translation is a small molecule selected from 4EGI-1, 4ElRCat, 4E2RCat, Allolaurinterol, Elisabatin A, Rocaglamide A, Silvestrol, Pateamine A, Hippuristanol and Ribavirin. In a preferred embodiment, the enhancer of IRES-mediated translation is 4EGI-1. In a preferred embodiment, the enhancer of IRES-mediated translation is 4ElRCat. In a preferred embodiment, the enhancer of IRES-mediated translation is 4E2RCat. In a preferred embodiment, the enhancer of IRES-mediated translation is Allolaurinterol. In a preferred embodiment, the enhancer of IRES-mediated translation is Elisabatin A. In a preferred embodiment, the enhancer of IRES- mediated translation is Rocaglamide A. In a preferred embodiment, the enhancer of IRES- mediated translation is Silvestrol. In a preferred embodiment, the enhancer of IRES-mediated translation is Pateamine A. In a preferred embodiment, the enhancer of IRES-mediated translation is Hippuristanol. In a preferred embodiment, the enhancer of IRES-mediated translation is Ribavirin.

[0087] In a preferred embodiment, the IRES mediated translation is IRES mediated circular RNA translation. In other words, the enhancer of IRES mediated translation enhance the translation of circular RNA operably linked to an IRES element.

[0088] In a preferred embodiment, the IRES element is a class 1 or class 2 IRES. In a preferred embodiment, the IRES element is a class 1 IRES. In a preferred embodiment, the IRES element is a class 2 IRES. In a preferred embodiment, the IRES element is selected from CVB3 and EMCV. In a preferred embodiment, the IRES element is CVB3. In a preferred embodiment, the IRES element is EMCV.

[0089] In a second aspect, the present invention provides a method for increasing translation of a circular RNA comprising the steps of

[0090] (a) providing an enhancer of IRES-mediated translation according to the first aspect of the invention; and

[0091] (b) administering said enhancer to cells comprising an IRES operably linked to the circular RNA to increase translation of the circular RNA.

[0092] The IRES is operably linked to the circular RNA so that the (at least one) protein encoded in the circular RNA can be translated from the circular RNA. The method of the second aspect of the invention can be used with any enhancer of IRES-mediated translation disclosed herein. In a preferred embodiment, the enhancer of IRES-mediated translation according to the first aspect is encoded in the same nucleic acid as the IRES operably linked to the circular RNA. In another preferred embodiment, the enhancer of IRES-mediated translation is administered together with the IRES operably linked to the circular RNA to the cells.

[0093] The method of the second aspect of the invention can for example be useful to enhance production of recombinant proteins, wherein the recombinant protein is encoded in a circular RNA comprising an IRES.

[0094] In a third aspect, the present invention provides a nucleic acid encoding the enhancer of IRES-mediated translation according to the first aspect of the invention. In a preferred embodiment, the nucleic acid is further encoding a circular RNA operably linked to an IRES.

[0095] In a fourth aspect, the present invention provides a vector encoding a nucleic acid according to the third aspect of the invention. In a preferred embodiment, the vector is an expression vector. Examples

[0096] The present invention provides an enhancer of IRES mediated translation. The enhancer utilizes inhibition of cap-dependent translation to increase IRES mediated translation.

[0097] Example 1: Small molecule inhibitor of cap-dependent translation

[0098] In an initial proof of concept experiment it was examined if blocking cap-dependent translation stimulates IRES-mediated circRNA expression. Therefore a small molecule inhibitor 4EGI-1 was used. 4EGI-1 disrupts the eIF4F complex and thereby inhibits capdependent translation in vitro. 4EGI-1 functions as a competitive binder interacting with eIF4E to prevent the eIF4E / eIF4G interaction. Additionally, 4EGI-1 inhibits the function of mTOR by blocking the phosphorylation of 4E-BP1.

[0099] In this proof of concept experiment A375 cells were first transfected with expression cassette plasmids encoding ICOSL circRNA (SEQ ID NO: 00015) and eGFP circRNA (SEQ ID NO: 0016) containing an internal CVB3 IRES. Twenty- four hours post-transfection cells were treated with 4EGI-1 at several concentrations (OpM-lOOpM). Forty-eight hours post 4EGI-1 treatment, protein and RNA expression was assessed by western blotting and qRT- PCR, respectively (Fig. 1).

[0100] 4EGI-1 treatment was found to increase protein expression from circICOSL and circEGFP in a dose dependent manner (Fig. 1A,C), whereas circICOSL and circEGFP RNA expression remained unaffected by 4EGL1 treatment at all dose-levels (Fig. 1B,D). Thus, demonstrating that the observed increase in protein expression is a consequence of increased translation not increased circRNA biogenesis.

[0101] Example 2: Phospho-mutant 4EBP1 co-expression stimulates circRNA-derived protein production

[0102] The 4EBP1 proteins are established regulators of cap-dependent translation, that competitively bind to eIF4E. The 4EBPl-eIF4E interaction inhibits eIF4E association with eIF4G and the formation of the eIF4F cap-dependent translation initiation machinery. However, phosphorylation of the 4EBP1 protein dissociates it’s interaction with eIF4E thus promoting cap-dependent translation. Several phosphorylation sites within 4EBP1 have been identified: Thr37, Thr46, Ser65, Thr70, Ser83, SerlOl, and Seri 12. The first five phosphorylation sites are phylogenetically conserved among all species, and phosphorylation of Thr37 and Thr46 serves as a priming event, which is followed by Thr70 phosphorylation and finally Ser65 phosphorylation.

[0103] In this experiment, two N-terminal FLAG tagged phosphorylation site-deficient 4EBP1 mutants were generated. One 4EBP1 mutant (4EBPl_dM; SEQ ID NO: 0004) contains T37A and T46A substitutions to disrupt the priming phosphorylation event. The second 4EBP1 mutant (4EBPl_DPhos; SEQ ID NO: 0006) contains T37A, T46A, S65A and T70A substitutions mimicking the fully hypo-phosphorylated state.

[0104] To examine the effect of 4EBP1 phospho-mutant expression on IRES mediated circRNA translation, A375 cells were first transfected with circEGFP encoding plasmids. Twenty- four hours post-transfection, plasmids encoding empty vector, 4EBPl_dM and 4EBPl_DPhos were transfected into circEGFP expressing cells. Twenty- four hours posttransfection, protein and RNA expression was assessed by western blotting and qRT-PCR, respectively (Fig. 2). FLAG-tagged 4EBP1 phospho-mutants expression was confirmed by western blot. Co-expression of 4EBPl_dM and 4EBPl_DPhos proteins enhanced eGFP protein expression (Fig. 2A). Additionally, qRT-PCR analysis found that circEGFP RNA levels decreased upon co-transfection with 4EBP1 phospho-mutants compared to empty vector control (Fig. 2B). This supports the results observed upon 4EGL1 treatment where inhibition of the cap-dependent translation machinery enhanced protein expression from an IRES- containing circRNA.

[0105] These results were recapitulated using another expression system to encode the 4EBP1 phospho-mutants. Here, expression of the 4EBP1 phospho-mutants was linked to an IRES element. Similar to the previous experiment, A375 cells were first transfected with circEGFP encoding plasmids and then twenty- four hours later transfected with the 4EBP1 encoding plasmids. Expression of the 4EBP1 phospho-mutants was confirmed by western blot. Again, eGFP protein expression was increased upon co-expression of the 4EBP1 mutants, while RNA levels remained unaffected.

[0106] Example 3: Knockdown of eIF4E enhances circRNA-derived protein production

[0107] Expanding on the observation that expression of the 4EBP1 phospho-mutant proteins enhance circRNA derived protein expression, the inventors examined if knockdown of eIF4E (i.e. the binding partner of the hypo-phosphorylated 4EBP1) would have a similar effect. Towards this goal, A375 cells were treated with control siRNAs and siRNAs targeting the capdependent translation initiation factor eIF4E (SEQ ID NO: 0018 or 0019). Forty-eight hours post-treatment A375 cells were co-transfected with control and eIF4E-targeted siRNAs and a plasmid encoding circEGFP. Forty-eight hours post-transfection RNA and protein was harvested and analyzed by qRT-PCR and western blot respectively. Western blot analysis showed that knockdown of eIF4E increased eGFP protein expression relative to control (Fig 3A). This suggests that inhibiting the eIF4E cap-dependent translation initiation factor is an attractive target to enhance IRES mediated translation, in particular IRES-mediated translation of circRNA.

[0108] Example 4: Effect of known inhibitors of cap-dependent translation The effects of a panel of viral proteins known to inhibit cap-dependent translation and / or mRNA stability on circRNA-derived protein production were examined. The specific inhibitory effect of each viral protein is described in Table 1 below.

[0109] Table 1 Specifically, in this experiment N-FLAG tagged versions of the poliovirus (PV) protease 2A and 3C, coxsackievirus B3 (CVB3) protease 2A, foot and mouth disease virus (FMDV) protease 3C, rotavirus (RV) NSP3, kaposi's sarcoma-associated herpesvirus (KSHV) Sox, simian vacuolating virus 40 (SV40) small T antigen (STA; SEQ ID NO: 0007) and the C- term of the small T antigen (STA C; SEQ ID NO: 0009) (Fig. 4A-B) and N-flag tagged decapping enzymes derived from the Vaccinia Virus (VACC) D9 (SEQ ID NO: 00011) and D10 (SEQ ID NO: 00013), as well as PAIP2 were co-transfected in A375 cells along with a plasmid encoding either circEGFP_CVB3 or Renilla mRNA. Additionally, as positive controls, the 4EBP1 phospho-mutants were also co-transfected with circEGFP or Renilla luciferase mRNA encoding plasmids.

[0110] Forty-eight hours post-transfection, circRNA derived eGFP protein expression and mRNA derived Renilla protein expression was assessed by western blotting. Western blot analysis showed that eGFP expression was increased upon expression of the SV40 small T antigen and C-term fragment (Fig 4A) and, upon co-expression of the N and C-flag tagged versions of the VACC D10 (Fig 4C). Additionally, co-expression of the 4EBP1 phospho- mutants also stimulated increased eGFP protein production.

[0111] For the other viral derived proteins tested no increase in eGFP expression relative to control was examined and, in some cases, eGFP expression was decreased (PV P2A and FMDV P3C) (Fig 4A &C). Renilla protein expression was either unaffected or depleted upon co-expression of these proteins (Fig 4A&C). The decrease in Renilla protein expression was particularly striking upon co-expression of the VACC D10 proteins (Fig 4C). However, this decrease can in part be explained by decreased RNA expression of the mRNA transcript (Fig 4D). Thus suggesting, that the VACC D10 proteins decap and destabilise mRNA transcripts. Additionally, as the circRNA molecules lack a 5 ’Cap, they are resistant to VACC_D10 and even show a modest increase in circRNA levels upon co-expression with VACC_D10 (Fig. 4D). The circRNA levels for the remaining proteins that had a beneficial effect on circRNA translation were not found to differ significantly upon co-transfection with the IRES boosters, suggesting the effect is translation dependent and not a consequence of increased circRNA expression.

[0112] Moreover, this effect was not limited to circRNAs encoding the CVB3 IRES, a similar effect was also observed for circRNAs encoding the EMCV IRES (Fig. 5; SEQ ID NO: 00017). Here, co-expression of either the 4EBPl_dM mutant and VACC D10 proteins had the greatest effect on circRNA mediated protein expression (Fig. 5A). No significant change was observed upon co-transfection of the 4EBPl_Phos mutant, however this protein was found to be expressed at a low level in this experiment (data not shown). Similar to the results obtained for the CVB3 IRES containing circRNAs, the circRNA levels were not significantly different between the 4EBPl_dM and the empty vector control, suggesting the increased protein expression is a consequence of changes in translation (Fig. 5B). Co-expression of the VACC D10 protein however did lead to an increase in circRNA expression. Additionally, the VACC D10 reduces Firefly mRNA levels, which correspond to a decrease in protein expression.

[0113] Summary of experimental evidence

[0114] Herein the inventors provide data demonstrating that inhibition of cap-dependent translation through different mechanisms can be utilized to enhance IRES-mediated translation from circRNAs. As IRES elements rely on cap-independent modes of translation they are unaffected by elements that inhibit cap-dependent translation. The inventors identified that interfering with specific stages of translation can positively impact IRES-mediated circRNA expression. Inhibition of the capping process and assembly of the cap-dependent translation initiation were identified as key processes that could be disrupted to enhance circRNA mediated protein production. Blocking of the eIF4E assembly into the trimeric translation initiation complex eIF4F, by co-expression of the 4EBP1 phosho-mutants or siRNA-mediated degradation of eIF4E was found to significantly enhance circRNA derived protein production. Moreover, inhibiting the assembly of this complex with 5 ’Capped mRNAs by expressing the Vaccinia Virus decapping enzyme DIO was also found to significantly enhance circRNA derived protein production.

[0115] Inhibition of other stages of the translation process were found not to impact or negatively impact circRNA derived protein expression such as expressing the Poliovirus P2A protease. This is of interest, as the expression of Poliovirus P2A protease has previously been reported to enhance translation from IRES elements. However, we observe that this is not the case for circRNAs containing such an IRES. Thus, suggesting that only disruption specific pathways allow for increased protein production.

Claims

Claims1. An enhancer of IRES mediated translation, wherein the enhancer is an inhibitor of capdependent translation.

2. The enhancer of IRES-mediated translation of claim 1, wherein the enhancer is an inhibitor of cap-dependent translation initiation, preferably selected from the group comprising: an inhibitor of the eIF4F complex and a decapping enzyme.

3. The enhancer of IRES-mediated translation of any one of the preceding claims, wherein the enhancer inhibits the eIF4F complex by interfering with(a) interaction of eIF4E with eIF4G; and / or(b) binding of eIF4E to capped mRNA.

4. The enhancer of IRES-mediated translation of any one of the preceding claims, wherein the enhancer directly interacts with eIF4E.

5. The enhancer of IRES-mediated translation of any one of the preceding claims, wherein the enhancer is selected from a small molecule, a protein and a nucleic acid.

6. The enhancer of IRES-mediated translation according to claim 5, wherein the protein is:(i) competing with eIF4G for binding to eIF4E, preferably the protein is a 4E- binding protein (4EBP);(ii) a decapping enzyme, preferably a decapping enzyme from Vaccinia Virus; or(iii) decreasing phosphorylation of a 4E-binding protein (4EBP), preferably SV40 small T-antigen.

7. The enhancer of IRES-mediated translation according to any one of the previous claims, wherein the enhancer is a 4EBP1 mutant protein, wherein at least one phosphorylation site selected from Thr37, Thr46, Ser65, Thr70, Ser83, SerlOl, and Seri 12 is inactivated, preferably at least Thr37 and Thr 46 are inactivated.

8. The enhancer of IRES-mediated translation according to claim 7, wherein the 4EBP1 mutant protein comprises at least at least substitutions T37A and T46A and optionally further comprises S65A and T70A substitutions.

9. The enhancer of IRES-mediated translation according to claim 5, wherein the nucleic acid is siRNA targeting at least one component of the eIF4F complex, preferably siRNA directed to eIF4E.

10. The enhancer of IRES-mediated translation according to claim 5, wherein the small molecule is selected from 4EGI-1, 4ElRCat, 4E2RCat, Allolaurinterol, Elisabatin A, Rocaglamide A, Silvestrol, Pateamine A, Hippuristanol, Ribavirin.

11. The enhancer of IRES-mediated translation of any one of the preceding claims, wherein the IRES mediated translation is IRES mediated circular RNA translation.

12. The enhancer of IRES-mediated translation according to claim 6, wherein the decapping enzyme is selected from Vaccinia Virus D9 and DIO.

13. A method for increasing translation of a circular RNA comprising the steps of(a) providing an enhancer of IRES-mediated translation according to any one of the previous claims; and(b) administering said enhancer to cells comprising a circular RNA operably linked to an IRES to increase translation of the circular RNA. a14. A nucleic acid encoding the enhancer of IRES-mediated translation according to any one of claims 1 to 12 and optionally further encoding a circular RNA operably linked to an IRES.

15. A vector encoding the nucleic acid of claim 14, preferably the vector is an expression vector.

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