SYSTEM AND METHOD FOR CELL-TYPE-SPECIFIC TRANSLATION OF RNA MOLECULES IN EUKARYOTES

Description

[0001] The present invention relates to methods and RNA constructs for the targeted translation of a polypeptide of interest in a eukaryotic target cell, as well as their use in therapeutic clinical applications. Background of the invention

[0002] The targeted introduction of specific molecules, for example medically and pharmacologically relevant proteins or peptides influencing cell vitality in cancer therapy, represents one of the greatest necessities but at the same time also a difficulty in modern cell biology that has not yet been satisfactorily solved.

[0003] Various techniques have been used to specifically release or activate molecules of interest in complex organisms such as humans. Two different approaches are of particular interest.

[0004] Based on carrier systems such as liposomes (D. Papahadjopoulos, M. Moscarello, EH Eylar, T. Isac, Biochim. Biophys. Acta. 1975, 401, 317), molecules (e.g. cancer therapeutics) are coupled to or embedded in these carrier systems. The thus functionalized carrier systems are then introduced into the body, whereby the introduction can cover either the entire body or just certain parts of the organism. Ideally, the carrier systems are designed in such a way that natural body conditions do not influence the stability between the carrier system and the molecule of interest, thus preventing the molecule from being released. External factors can now destabilize the carrier systems and thereby induce the release and thus activity of the incorporated molecules. External factors for release include local heating of certain body regions (hyperthermia approach) as well as light orpH-induced cleavage of specific bonds between the carrier system and the molecule has been used. The principles of all these systems have been summarized by Qiu and Park (Advanced Drug Delivery Reviews 53 (2001) 321-339 Environment-sensitive hydrogels for drug delivery) and Shamay and colleagues (Biomaterials, Light induced drug delivery into cancer cells 2011 32:1377-86). A particular problem with all of these methods, however, is that the functional molecules must be introduced into the entire body in high concentrations, which can often lead to side effects and non-specific release. Furthermore, these methods are site-specific but not cell-type-specific, meaning that cytotoxic drugs, for example, can damage not only the intended cancer cells but also the surrounding tissue, or can be rapidly distributed throughout the body from the site of release. High doses are therefore required here as well.

[0005] As a second method for the targeted release of molecules, systems are usually used in which the molecules of interest are coupled, either directly or via carrier systems, to ligands that exhibit high specificity for certain surface molecules of defined cell types. Highly expressed surface receptors of cancer cells, such as GPRCs or folate receptors, are thus bound via the ligands (Lappano R, Maggiolini M., Nat Rev Drug Discov. 2011, 10:47-60, G protein-coupled receptors: novel targets for drug discovery in cancer; Sudimack J, Lee RJ., Adv Drug Deliv Rev. 2000, 41:147-62. Targeted drug delivery via the folate receptor).After the ligands bind to the surface receptors, bound molecules can either exert their activity directly (e.g., radiopharmaceuticals or MRI contrast reagents) or exert their function after phagocytic uptake into the cells (e.g., chemotherapeutics or DNA vectors). However, this method also has major disadvantages, which lie, on the one hand, in the relatively high overall concentration throughout the body system, and, on the other hand, in the fact that most surface receptors do not exhibit absolute specificity for individual cell types or disease patterns, and thus coupling to such receptors can be associated with increased side effects in healthy tissue. Furthermore, a large number of specific receptors do not induce phagocytic uptake of the bound carrier systems after coupling, so that they remain outside the cells.

[0006] The vast majority of biotechnologically, pharmacologically, and medically relevant molecules for targeted delivery into defined cell types / tissues are peptides or proteins, typically encoded at the DNA level. After introduction of these DNA molecules, they are transported into the cell nucleus and subsequently, via a highly conserved mechanism, first transcribed into RNA molecules (transcription), posttranscriptionally modified, and then transported out of the nucleus to be transcribed into amino acid sequences within the cytoplasm by a highly conserved mechanism (translation). Both the transcription and translation mechanisms are fundamentally identical in all eukaryotic organisms and cell types.This means that, albeit with varying efficiency, each DNA sequence encoding a peptide produces the same primary product in every cell, which can subsequently be further modified. The entire process of transcription and translation is called expression. Some RNA molecules remain untranslated and perform an independent function.

[0007] Selective, targeted expression of DNA sequences is possible when the coding region of a DNA segment is regulated by cell type- or organ-specific promoter and enhancer sequences as well as by the degree of differentiation and maturation. Such sequences regulate the binding of the transcription apparatus and thus cause its activation only under defined environmental conditions. The disadvantage of this type of targeted expression of DNA constructs, however, is that 1) DNA constructs are very difficult to introduce with high efficiency and homogeneously into all cells within an organism or tissue, 2) DNA constructs must always be transported into the cell nucleus for subsequent transcription, where they interact with the natural genetic material, which can lead to mutations and subsequent diseases (e.g.cancer) and 3) organ-specific promoter regions are often very large and thus not suitable for use in externally introduced DNA constructs.

[0008] As an alternative to using DNA constructs to express amino acid-based molecules, the transcription step that takes place in the cell nucleus can be bypassed by directly introducing RNA molecules into cells or cell tissue. Work by various research groups has shown (for a review see: Van Tendeloo VF, Ponsaerts P, Berneman ZN. mRNA-based gene transfer as a tool for gene and cell therapy. Curr Opin Mol Ther. 2007;9:423-431) that the introduction of RNA molecules occurs highly efficiently and rapidly without generating significant levels of stress in the cells. Introduced RNA molecules remain in the cytoplasm of the cell and thus cannot negatively interact with the genome within the cell nucleus. Through conserved translation processes, introduced mRNA molecules are converted into proteins or peptides in the same way as endogenous (naturally occurring) mRNA molecules.This requires certain sequence motifs within an mRNA molecule, which can be read in detail in textbooks (e.g. Alberts et al., Molecular Biology of the Cell, 2011, Wiley-Blackwell Verlag) and simplified in . Fig. 1 mRNA molecules typically comprise the following units: 1: Cap: The cap structure is a chemical modification of mRNA molecules in eukaryotes that drastically increases the stability of the RNA and is important for the transport of RNA from the nucleus into the cytoplasm and the subsequent translation of mRNAs by the ribosomes. It is usually a modified guanine nucleotide that is attached to the head end of the RNA via a rare 5'-5' phosphodiester bond during gene transcription. 2: Untranslated 5' region: Represents a nucleotide sequence upstream of the coding sequence. This region begins at the transcription start site and ends directly before the translation start codon. Regulatory sequences can be located within this sequence, which, for example, influence the stability of the mRNA via secondary structures or act as binding sites for proteins. A ribosomal binding site is also typically present.3: Coding sequence: The coding sequence contains the information for the protein to be produced by the mRNA. It begins directly with the translation start codon and ends with the translation stop codon. The nucleotide sequence of the coding sequence is determined by the amino acid sequence of the encoded protein within the triplet code. Depending on the triplet used, the expression rate and stability of the mRNA can be influenced. 4: Untranslated 3' region: Represents the region immediately following the protein coding region after the translation stop codon. It encompasses the entire region up to the start of polyadenylation. Regulatory sequences that are specifically important for the polyadenylation of the RNA as well as for its stability and transport can also be present within the untranslated 3' region.5: polyA tail: Polyadenylation occurs by attaching adenine nucleotides to the 3' end of the mRNA based on post-transcriptional modification of the mRNA by poly(A) polymerase. The polyA tail contributes significantly to the stability of the mRNA through its length, as well as through the binding of proteins to this sequence and the interaction of the CAP with the polyA tail. WO2015042184 describes RNA constructs that bind and / or inhibit non-coding RNA, such as miRNA.

[0009] However, the use of externally introduced RNA molecules can be disadvantageous for certain applications because i) RNA molecules typically have a limited lifespan, meaning that protein formation only occurs over a limited period of time. Since the basic mechanism of translation remains identical in all eukaryotes and their cell types, ii) targeted, i.e., organ- or cell-type-specific expression of mRNA molecules introduced into the entire organism has not yet been achieved.

[0010] New research results (Quabium and Krupp, Synthetic mRNAs for manipulating cellular phenotypes: an overview (2015) New Biotechnology, 32:229-235) now enable the very efficient chemical modification of mRNA molecules at different levels in order to regulate the lifespan listed under point i) and, if necessary, to significantly increase it. Such modifications include the a) Use of chemically modified cap sequences based on the naturally occurring cap m7G5'pppN. Examples of this can be found in Jamielity et al. (Synthetic mRNA cap analogs with a modified triphosphate bridge - synthesis, applications and prospects (2010) New. J. Chem 34:829-844). Such modifications increase translation efficiency through effective binding of subunits of the translation apparatus. They also improve the stability of mRNA molecules, presumably because nucleases are less able to attack the mRNA from the 5-terminus. b) Use of stable 5' and 3' untranslated regions. Typically, stability can be significantly influenced by using suitable nucleotide sequences. The attachment of binding partners and the formation of secondary structures through base pairing are the main reasons for stabilization. c) Use of chemically modified nucleotides.Nucleotides such as 5-methylcytidine or pseudouridine primarily influence the binding of endonucleases and thus prevent the rapid degradation of mRNA molecules. d) Use of efficient polyadenylation sequences in the untranslated 3' region to achieve the longest possible polyadenylation.

[0011] The object of the present invention is, among other things, to provide methods and RNA constructs that allow a defined expression of mRNA in certain cell types. Brief description of the invention

[0012] A first aspect of the present invention relates to a method for the targeted translation of a polypeptide of interest in a eukaryotic target cell, characterized by introducing an RNA construct into the target cell, wherein the RNA construct comprises at least the following elements in the 5' to 3' direction: (a) an anticodogenic element (a) that is complementary to at least a portion of an mRNA of a gene expressed by the target cell; (b) a blocking element (b) that interacts with at least a portion of the 3' translation initiator element (d); (c) a stabilizing element (c); (d) a translation initiator element (d) that has an internal ribosomal entry site (IRES) sequence; (e) a sequence coding for the polypeptide of interest (e), wherein in a eukaryotic non-target cell the translation of the polypeptide of interest is prevented by base pairing of the blocking element (b) with the translation initiator element (d) and in a eukaryotic target cell the translation of the encoded polypeptide occurs due to processing of the RNA construct induced by the interaction of the anticodogenic element with the mRNA expressed by the target cell.

[0013] A further aspect of the present invention is directed to an RNA construct for the targeted translation of a polypeptide of interest in a eukaryotic target cell, wherein the RNA construct comprises at least the following elements in the 5' to 3' direction: (a) an anticodogenic element (a) which is complementary to at least a portion of an RNA of a DNA segment expressed by the target cell or which induces cell-type-specific degradation; (b) a blocking element (b) which is complementary to at least a portion of the 3' translation initiator element (d) or which otherwise inhibits it; (c) a stabilizing element (c) which restricts the target cell-specific degradation of the RNA to a portion thereof; (d) a translation initiator element (d) which functions independently of the 5' cap and is only active in target cells; wherein the translation initiator element (d) has an internal ribosomal entry site (IRES) sequence; (e) a sequence (e) coding for the polypeptide of interest; wherein in a eukaryotic non-target cell the translation of the polypeptide of interest is prevented by base pairing of the blocking element (b) with the translation initiator element (d) and in a eukaryotic target cell the translation of the encoded polypeptide occurs due to an interaction of the anticodogenic element (a) with an RNA expressed by the target cell, a specific processing of the RNA construct whereby the translation initiator element (d) is activated.

[0014] Further aspects of the present invention are the use of the methods and RNA constructs described herein in therapeutic clinical applications, in neoplasms, underlying immunological diseases, and metabolic disorders. Furthermore, the methods and RNA constructs described herein are suitable for specifically expressing the necessary components for gene editing in a cell type and thus for treating germline diseases.

[0015] In addition to these typical applications, adult mammalian stem cells (iPS, multipotent, totipotent, or pluripotent) and their derived tissues can also serve as target cells in vitro. The resulting tissues or single cells can then be used both in vivo in humans and in drug development. Short description of the figures

[0016] Figure 1: Schematic representation of an mRNA molecule. All segments of an mRNA can have a regulatory function for the stability of the molecule. Figure 2: Schematic representation of an embodiment of an RNA construct of the present invention for the selective expression of target sequences in specific cell types after organ- or organism-wide introduction of the mRNA molecule. Figure 3: Schematic representation of another embodiment of an RNA construct of the present invention for the selective expression of target sequences in specific cell types, where 1 = CAP / modifications, 2 = 5'-UTR, 3 = anticodogenic fragment, 4 = linker_IRES blocker_linker, 5 = stabilizing element, 6 = YOURS, 7 = sequence of interest, 8 and 9 = 3'-UTR and polyA.Figure 4: A) Schematic representation of an embodiment of an RNA construct of the present invention before and after processing by anticodogenic interaction between element 3 and cell-type-specific RNA. Upon binding to target RNA, the anticodogenic sequence element leads to the degradation of motifs 1-4 by established mechanisms. B) Calculated secondary structure of an exemplary functional IRES sequence (6) from HCV (HCV IRES 1b). Figure 5: Primary IRES nucleic acid sequence (HCV IRES 1b); position 6 in . Fig.4 : SEQ ID No. 1. The corresponding functional secondary structure is shown in Fig. 4Figure 6: A) Schematic representation of an embodiment of an RNA construct of the present invention before and after processing by anticodogenic interaction between element 3 and cell-type-specific RNA. The anticodogenic sequence element leads to the degradation of motifs 1-4 upon binding to target RNA by established mechanisms. B) Schematic representation of the secondary structure of an exemplary IRES sequence (6) from HCV (HCV IRES 1b) with 3 stab. elements (5). Figure 7: Primary IRES sequence (position 6 in Fig.6 ) with 3 stabilizing elements (position 5 in Fig. 6 ): SEQ ID No. 2. The corresponding functional secondary structure is shown in Fig. 6Figure 8: A) Schematic representation of an embodiment of an RNA construct of the present invention before and after processing by anticodogenic interaction between element 3 and cell-type-specific RNA. The anticodogenic sequence element, upon binding to target RNA, leads to the degradation of motifs 1-4 by established mechanisms. B) Schematic representation of the secondary structure of an exemplary IRES sequence (6) from HCV (HCV IRES 1b) with 3 stab. elements (5) and linker (4a). Figure 9: Primary IRES sequence (position 6 in Fig.8 ) with 3 stabilizing elements (position 5 in Fig. 8 , underlined) and linker (position 4a in Figure 8 , boxed): SEQ ID No. 3. The corresponding functional secondary structure is shown in Fig. 8Figure 10: A) Schematic representation of an embodiment of an RNA construct of the present invention before and after processing by anticodogenic interaction between element 3 and cell-type-specific RNA. The anticodogenic sequence element leads to the degradation of motifs 1-4 upon binding to target RNA by established mechanisms. B) Schematic representation of the secondary structure of an exemplary IRES sequence (6) from HCV (HCV IRES 1b) with 3 stab. elements (5), linker (4a), and blocker (4b). Figure 11: Primary IRES sequence (position 6 in Fig.10 ) with 3 stabilizing elements (position 5 in Fig. 10 , underlined), linker (position 4a in Figure 8 , boxed) and blocker (4b, bold): SEQ ID No. 4. The corresponding secondary structure is shown in Fig. 10Figure 12: Example of a nucleotide sequence of the functional system for the selective expression of target sequences in defined cell types. Figure 13: Example of a nucleotide sequence of the functional system with an alternative antisense sequence for the selective expression of target sequences in defined cell types. Figure 14: Example of a nucleotide sequence of the functional system with an alternative antisense sequence and an alternative blocker sequence for the selective expression of target sequences in defined cell types. Figure 15: Microscopic images of the cell type-dependent, blocker-IRES-controlled, selective expression of GFP. Figure 16: Tables for the quantitative evaluation of the functional system for the selective expression of target genes.

[0017] In the following, the invention is explained in more detail without limiting the general inventive concept using exemplary embodiments with reference to the figures.

[0018] The scope of the invention is defined by the appended claims. Detailed description of the invention

[0019] The basic structure of the RNA molecule includes stabilizing structures, a regulatory region, and a functional region that encodes the sequence of interest. The regulatory structure acts as a constitutive inhibitor of the expression of the sequence of interest. This inhibition is only released when specific binding induces the selective degradation of the regulatory region, thus enabling the expression of the sequence of interest.

[0020] The invention comprises the method for constructing suitable RNA sequences which, after introduction into any cell type, make it possible to induce the functionality of a target sequence or of a resulting amino acid-based structure only in defined cell types and to prevent it in all others.

[0021] To solve the problem of defined expression of mRNA molecules in specific cell types after introduction into the entire organism or parts thereof, different sequence motifs are combined in this patent application in such a way that functional translation of the sequence of interest is only possible in desired cell types, while in other cell types this does not occur. By adapting the sequence motifs, the cell type-dependent interplay between activation and inactivation can be adapted or even completely reversed. In general, the system is constructed in such a way that upstream sequences in the 5' region regulate the stability of the system and simultaneously inhibit or generally regulate sequence motifs of interest in the 3' region. Inhibited sequences within the 3' region are, in particular, internal ribosomal entry sites (IRES) sequences, which can serve as secondary translation initiators.Inhibition via base pairing within the mRNA molecule is maintained permanently until the mRNA encounters cell-type-specific, endogenous mRNA molecules in suitable target cells. These molecules form antisense-active base pairs with sequences in the 5' region of the introduced mRNA, thereby inducing the degradation of the front region of the introduced mRNA. This removes the inhibition of the IRES and enables expression of the target sequence. In detail, the construction of the cell-type-specific, controllable mRNA molecules occurs according to the procedure described in . Fig. 2 The scheme shown is explained in more detail below. Depending on the need, individual sequence motifs can be omitted, their order swapped, replaced by other motifs, or supplemented by additional motifs.

[0022] Fig. 2. Shows a schematic representation of an mRNA molecule for the selective expression of target sequences in specific cell types after organ- or organism-wide introduction of the mRNA molecule. 1) Optional use of a CAP sequence. This sequence can represent a natural 7-methylguanosine (m7G) cap or contain chemical or other modifications. The CAP sequence serves to regulate the stability and translation efficiency of the primary sequence. For certain applications, such as the development of RNA molecules or RNA fragments with low stability, the CAP sequence can be omitted. 2) Use of a 5'-untranslated sequence. This sequence primarily serves to regulate the stability (half-life) of the introduced mRNA. Nucleotide sequences can either be synthetic or taken from sequences of known genes. Depending on the required stability, additional sequences can be integrated into this region, individually or in combination, to regulate stability and translation rate.Examples would be G-quadruplexes (high mRNA stability with a simultaneous low translation rate) or binding sites for endonucleases (reduced mRNA stability). The length of the 5' untranslated sequence is freely selectable and can even be zero if necessary. 3) Following the 5' untranslated sequence, the actual regulatory cassette for the cell-type-specific expression of artificially introduced mRNA molecules starts. The starting point is an antisense or RNAi-active anticodogenic sequence within the introduced mRNA against a gene specifically expressed in a desired cell type. If the mRNA construct is introduced into a cell in which the cell-type-specific gene is not transcribed, no interaction occurs. However, if such a sense mRNA is present, base pairing occurs between the sense and antisense strands, which induces subsequent degradation of both the sense and antisense strands.The degradation itself is initiated and carried out by protein complexes conserved in all cells, with the enzyme complex RISC (RNA-induced silencing complex) and the ribonucleases Dicer and Drosha playing a particularly important role (Sontheimer EJ (2005). "Assembly and function of RNA silencing complexes". Nature Reviews Molecular Cell Biology. 6: 127-138). Depending on surrounding sequences, the degradation can encompass the entire introduced mRNA strand or be restricted by additional sequences.

[0023] Cell type or organ-specific protein expression patterns are known as state of the art based on modern transcriptome or proteome analyses for almost every cell type or tissue and can easily serve as a basis for pre-selection (see, for example, Ko et al. PNAS, 2013, 110:3095-3100 or Whitfield et al. PNAS, 2003, 100:12319-12324).

[0024] To identify suitable anticodogenic elements (3) with the best possible antisense function, different parameters can be taken into account. For example, see: Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.

[0025] According to the invention, the following parameters should play a role: Anticodogenic sequence preferably 50 to 100 nt downstream from the start codon Search for sequence motifs AA(N 19 )TT or NA(N 21 ), or NAR(N 17 )YNN, with N = any nucleotide, R = purine and Y = pyrimidine Anticodogenic sequence with G+C content of 35-60% if possible Avoid four or more identical nucleotides in a row Avoid sequence motifs with high homology to other genes

[0026] 4) Following the antisense / RNAi active sequence is a sequence capable of base pairing or other interactions with the IRES sequence located in the 3' region of the construct, thus acting as an IRES blocker. Since IRES sequences themselves are also capable of exerting their function as CAP-independent translation initiators primarily or exclusively through specifically constructed secondary structures, the additional base pairings between the IRES blocker and the IRES sequence alter these in such a way that no IRES-dependent translation initiation occurs as long as the IRES blocker is present. The IRES blockage occurs when the introduced mRNA is of its full length. Since defined induced changes in the length of the mRNA only occur when the specific antisense / RNAi sequence binds to sense mRNA, IRES activity is absent in all cells that do not have a suitable sense mRNA.The presence of this sense mRNA in specific cell types, however, leads to antisense-sense binding and thus to the induction of the degradation of the introduced mRNA, which also affects the sequence of the IRES blocker and thus loses its blocking effect on the secondary structure formation of the IRES sequence. The IRES blocker sequence is characterized by the fact that it i) contains anticodogenic segments of the IRES sequence, ii) binds to one or more sequence segments of the IRES sequence, iii) can have spacer sequences between the segments anticodogenic to the IRES sequence, which can have different compositions in length and nucleotide composition, iv) preferentially binds to stem sequences or loop sequences of the IRES in order to change their secondary structure, v) that the sequences have a variable length of nucleotides anti-codogenic to the IRES without typically exhibiting antisense or RNAi activity themselves.Changes in the secondary structure of the IRES caused by the blocker can be minimal, effectively inhibiting IRES function and allowing refolding of the IRES sequence upon degradation of the blocker. To ensure optimal binding between the IRES blocker and the IRES, the IRES blocker sequence can be surrounded by linker sequences of variable length to both upstream and downstream sequence motifs.

[0027] 5) In order to restrict the complete degradation of the introduced mRNA to specific regions after binding of sense and antisense / RNAi mRNA motifs, mRNA sequence motifs must be incorporated that interrupt the degradation due to binding partners or developed secondary structures. Such sequence motifs can include hairpin loops or stem loops. They can be used in single or multiple arrangements in both the 5' and 3' directions for sense-antisense pairing. The use of sequence motifs that are typically stabilized by secondary structures or binding partners, in combination with the specific antisense / RNAi cassette in target cells with sense pairing, interrupts mRNA degradation and thus stabilizes the RNA motifs located 3' to this sequence.Here, the stable secondary structures take over the function of the CAP with regard to stabilization, but not with regard to translation initiation. In cells where no sense-antisense pairing occurs, the total length of the mRNA is maintained, and the loop structures remain without significant function. Stable mRNA secondary structures can be linked to neighboring sequence motifs with or without connecting sequences (linkers).

[0028] 6) Since the CAP sequence required for translation initiation is cleaved off in target cells, a CAP-independent ribosomal binding site must be integrated into the sequence behind the stable mRNA secondary structures to initiate peptide or protein formation. For this purpose, an IRES sequence acting as a translation initiator is incorporated in the 3' direction. Due to its developed secondary structure, this IRES sequence typically enables the direct binding of ribosomal subunits. IRES sequences can be cellular, viral, or synthetic. Due to IRES blocker sequences matching the IRES sequence in the same full-length mRNA construct, which prevent the formation of the IRES secondary structure required for translation initiation through base pairing, the IRES sequence is inactive in non-target cells and thus in the full-length mRNA present.In target cells, however, the IRES blocker sequence is degraded through sense-antisense / RNAi interactions, whereby the functional IRES secondary structure is formed through energetically driven processes and can function for the translation of subsequent coding sequences. The IRES sequence can be incorporated with or without linker sequences to surrounding sequence motifs.

[0029] 7) Coding sequences can be inserted 3' to the inserted IRES structure, which are transcribed into peptides by translation. Translation of these sequence motifs occurs exclusively in cells in which the IRES sequence is active, i.e., in target cells in which endogenous sense sequences are also transcribed for the antisense / RNAi motif within the inserted mRNA. RNA sequences to be translated can encode, for example, therapeutically active peptides.

[0030] 8) Following the defined translatable reading frame, further sequences in the 3' direction are required, which in turn ensure the stability of both the full-length mRNA and the mRNA truncated in target cells. These sequences include both the untranslated 3' region and the polyA tail, which is also characteristic of almost every endogenous mRNA. Depending on the need, endogenous or synthetic sequence motifs can be used. Depending on the desired stability of the mRNA construct, this can be specifically adjusted by selecting the appropriate sequence motif. Of particular importance here are i) formed secondary structures within the motif, ii) possible binding sites for stabilizing / destabilizing factors, and iii) the length of the polyA tail.

[0031] Based on this system, the selective, adjustable expression of target sequences is possible for the first time, even when mRNA molecules are introduced into the entire organism.

[0032] The RNA constructs of the present invention can be introduced into a target cell in a variety of ways. They can enter the cell via transfection, membrane fusion, electroporation, or as a retroviral vector. For example, WO 02 / 44321 discloses the introduction of a foreign target gene into a cell.

[0033] In a particular embodiment of the present invention, the interaction of the anticodogenic element (a) with the mRNA expressed by the target cell leads to degradation or inactivation of the blocking element (b), so that translation of the polypeptide of interest can occur. In a further embodiment, the degradation of the mRNA is stopped at the stabilizing element (c).

[0034] In further preferred embodiments of the invention, the anticodogenic element (a) has one or more of the following features and has the following function or functions after introduction into the target cell: (i) Binding to an RNA sequence specific for the target cell. (ii) Formation of double-stranded RNA hybrids of freely adjustable length. (iii) Induction of cellular degradation of the introduced RNA construct in the region of the double-stranded RNA hybrid.

[0035] In further preferred embodiments of the invention, the blocking element (b) has one or more of the following features: (i) the function of the translation initiator element is suppressed. (ii) the blocking element can be degraded or inactivated under suitable conditions. (iii) the suppressive function on the translation initiator element is reversible after inactivation or degradation of the blocking element. (iv) the blocking element exerts its function by base pairing in or near the sequence of the translation initiator element, thereby altering its secondary structure. (v) the blocking element exerts its function by causing other molecules to directly or indirectly inhibit the translation initiator element.

[0036] In further preferred embodiments of the invention, the stabilizing element (c) has one or more of the following features: (i) Nucleotide sequence that prevents the degradation of RNA in the 3' direction by exo- and / or endonucleases. (ii) Nucleotide sequence characterized by the formation of stabilizing secondary structures, such as stem loops. (iii) Nucleotide sequence characterized by the binding of stabilizing elements such as peptides or proteins to the RNA. (iv) Nucleotide sequence characterized by the binding of molecules to the RNA and chemically modifying the RNA 5' end.

[0037] In further preferred embodiments of the invention, the translation initiator element (d) has one or more of the following features: (i) Cell type-dependent, selective inhibition of the translation initiation function (ii) Functionality exclusively upon degradation or inactivation of the components of the regulatory cassette (elements a and b) (iii) Functionality exclusively in target cells, while in all other cell types the translation initiator element is inactive. (iv) Induction of translation independent of the cap structure in the 5' region of the starting RNA construct. (v) Induction of translation by direct or indirect binding of ribosomal subunits or complete ribosomes (e.g., IRES sequences). (vi) Induction of translation by formation or release of specific secondary structures after removal of inhibition by the blocking element. (vii) Induction of translation by attachment of specific molecules or chemical modification after removal of inhibition by the blocking element.

[0038] In particularly preferred embodiments of the present invention, the RNA construct has a combination of anticodogenic and IRES-dependent expression regulation.

[0039] The degradation of the blocking element can occur, for example, via the system of antisense- or siRNA-dependent mRNA degradation. The phenomenon, known in the literature as "RNA interference" (RNAi), is based on the interaction of short RNA molecules (siRNA, small interfering RNA) with messenger RNA (mRNA) in the cell (Reference: Fire A., Xu S., Montgomery MK, Kostas SA, Driver SE, Mello CC, Nature Feb 19, 1998; 391 (6669):744-745). A complex mechanism controlled by enzymes (for example, the DICER and RISC complexes) leads to the degradation of the mRNA. In addition to siRNA, other small RNA species have been discovered, such as microRNAs (miRNA) or short hairpin RNAs (shRNA), which can also inhibit protein expression through related mechanisms.

[0040] The translation initiation of the RNA construct in the target cell, initiated by the degradation and / or inactivation of the blocking element, is a complex process involving the concerted interaction of numerous factors (Pain, VM, 1996, Eur. J. Biochem. 236, 747-771). For most mRNAs, the first step is the recruitment of 40S ribosomal subunits to the RNA at or near its 5' end ( Fig. 1 ). The association of 40S to the mRNA is greatly facilitated by the cap-binding complex eIF4F. eIF4F consists of three subunits: the RNA helicase eIF4A, the cap-binding protein eIF4E, and the multiadaptor protein eIF4G, which acts as a framework for the proteins in the complex and has binding sites for eIF4E, eIF4a, eIF3, and a poly(A)-binding protein.

[0041] Infection of cells with a variety of RNA viruses leads to the selective inhibition of translation of host mRNA but not of the virus. For example, infection of cells with poliovirus, a cytoplasmic RNA virus, leads to the modification of several translation initiation factors. In particular, proteolysis of both forms of elF4G, elF4Gl, and elF4Gl (Gradi et al. 1998, Proc. Nat. Acad. Sci. USA 95, 11089-11094), by virus-encoded proteases leads to the inhibition of translation of most capped cellular mRNAs. In contrast, translation of polioviral mRNAs containing a specific 450-nucleotide IRES in the 5' UTR, which can recruit 40S subunits in the absence of intact eIF4F, is not inhibited (Jang et al. 1988 J. Virol. 62, 2363-2643). IRES elements have been found in picornaviral, flaviviral, pestiviral, retroviral, lentiviral, insect viral RNA, and animal cellular RNA.Overviews of known IRES sequence motifs represent the state of the art (presented as an overview at: http: / / www.iresite.org (as of February 1, 2017). Animal mRNAs containing IRES can recruit ribosomal subunits both via their 5' cap end and via their IRES elements. This enables translation even under conditions in which cap-dependent translation is reduced, such as during viral infection, during the G2 / M phase of the cell cycle, apoptosis, or stress conditions (Johannes et al. (1999), Proc. Natl. Acad. Sci. USA 96, 13118-13123; Cornelis et al. (2000), molecular Cell 5, 597-605; Pyronnet et al. (2000), molecular Cell 5, 607-616; Stein et al., 1998, Mol. and Cell. Biol. 18, 3112-3119; Holcik et al., 2000, Oncogene 19, 4174-4177; Stoneley et al., 2000, mol. and Cell. Biol. 20, 1162-1169).Up to 3% of animal cellular mRNAs are translated when the concentration of the cap-binding complex elF4F is reduced (Johannes et al., 1999).

[0042] For the RNA constructs of the present invention, a variety of different IRES elements can be used as translation initiator elements. For example, the prior art describes IRES elements used for cap-independent expression of foreign genes in linear multicistronic mRNA in animal cells (US 6,060,273; US 6,114,146; US 5,358,856; US 6,096,505; US 171,821; US ​​5,766,903), in plant cells (WO 98 / 54342), and, generally, in eukaryotic cells (US 171,821; US ​​5,766,903; US 5,925,565; US 6,114,146).

[0043] For example, the secondary structure of the IRES element remains unaffected and thus functional and is only changed (= inactivated) by a suitable choice of the blocking element (b).

[0044] In further preferred embodiments of the invention, the RNA construct has one or more of the following optional features: (i) 5'-cap sequence to increase the stability of the entire mRNA construct (ii) 5'-untranslated region to regulate the stability of the entire mRNA construct and to suppress 5'-cap-induced translation (e.g. incorporation of quadruplex sequences) (iii) 3'-untranslated region to increase the stability of the entire mRNA construct and of the processed mRNA partial fragment in target cells. (iv) Poly-A region to increase the stability of the entire mRNA construct and of the processed mRNA partial fragment in target cells. (v) All elements of the entire mRNA construct from claims 1 to 8 can contain linker sequences of any length within the element itself or between the individual elements. Linker sequences can be non-functional and serve purely to spatially separate the individual elements, or they can integrate additional functions into the system. Examples of implementation: 1.) Nucleotide sequence of the functional system for the selective expression of target sequences in defined cell types (Figure 12).

[0045] Using standard molecular biological procedures (PCR, ligation and transformation), a short sequence of the Keratin13 gene (marked above with short KERATIN ) of the human genome as antisense with a so-called LOOP sequence. The LOOP -Sequence includes Left -Sequences that serve the flexibility of the construct, a Blocker sequence, as well as nucleotide sequences that form stable secondary structures ( LOOPs ) . While the Blocker -sequence is capable of dealing with sequence sections of the IRES to interact through base pairing and thereby change their secondary structure, the secondary structures of the LOOPs an interruption of exo-nuclease activity (see Chapman et al. eLife, 2014,3:e01892). The following sequence segments represent the IRESof the Encephalomyocarditis Virus (Bochkov and Palmenberg, BioTechniques, 2006, 41:283-292) and the coding sequence of the green fluorescent protein (eGFP) The IRES allowed if the Blocker sequence the translation of subsequent sequences - in this case the eGFP, which was chosen here for reasons of easy verification. Blocker sequence only occurs if the antisense sequence ( short KERATIN ) there is a complementary sense sequence for binding in the selected cell type. 2.) Nucleotide sequence of the functional system with alternative antisense sequence for the selective expression of target sequences in defined cell types (Figure 13).

[0046] Using standard molecular biological procedures (PCR, ligation and transformation), a long sequence of the Keratin13 gene (marked above with KERATIN ) of the human genome as antisense with a so-called LOOP sequence. The LOOP -Sequence includes Left-Sequences that serve the flexibility of the construct, a blocker sequence, as well as nucleotide sequences that form stable secondary structures ( LOOPs ). While the Blocker -sequence is capable of dealing with sequence sections of the IRES to interact through base pairing and thereby change their secondary structure, the secondary structures of the LOOPs an interruption of exo-nuclease activity (see Chapman et al. eLife, 2014,3:e01892). The following sequence segments represent the IRES of the Encephalomyocarditis Virus (Bochkov and Palmenberg, BioTechniques, 2006, 41:283-292) and the coding sequence of the green fluorescent protein (eGFP) The IRES allowed if the Blocker sequence the translation of subsequent sequences - in this case the eGFP , which was chosen here for reasons of easy verification. Blocker- Sequence only occurs if the antisense sequence( KERATIN ) there is a complementary sense sequence for binding in the selected cell type. 3.) Nucleotide sequence of the functional system with alternative antisense sequence and alternative blocker sequence for the selective expression of target sequences in defined cell types (Figure 14).

[0047] Using standard molecular biological procedures (PCR, ligation and transformation), a long sequence of the Keratin13 gene (marked above with KERATIN ) of the human genome as antisense with a so-called LOOP sequence. The LOOP -Sequence includes Left -Sequences that serve the flexibility of the construct, a Blocker sequence, as well as nucleotide sequences that form stable secondary structures ( LOOPs ). While the Blocker -sequence is capable of dealing with sequence sections of the IRES to interact through base pairing and thereby change their secondary structure, the secondary structures of the LOOPs an interruption of exo-nuclease activity (see Chapman et al. eLife, 2014,3:e01892). The following sequence segments represent the IRESof the Encephalomyocarditis Virus (Bochkov and Palmenberg, BioTechniques, 2006, 41:283-292) and the coding sequence of the green fluorescent protein (eGFP) The IRES allowed if the Blocker sequence the translation of subsequent sequences - in this case the eGFP, which was chosen here for reasons of easy verification. Blocker- Sequence only occurs if the antisense sequence ( KERAT IN ) there is a complementary sense sequence for binding in the selected cell type. 4.) Cell type-dependent, blocker-IRES-controlled, selective expression of GFP (Figure 15).

[0048] Mouse epidermal cells (keratinocytes) with (WT) and without (knockout keratin13 mutant = KO) keratin13 mRNA were treated with GFP expression plasmids as a positive control. After 24 h, the cells were examined by light microscopy (gray) and fluorescence microscopy (green). As a result of the transfer, a uniformly high level of GFP expression was observed in both cell lines.

[0049] By using the Fig. 1 In the constructs generated by 2 (clone 174) and 2 (clone 215), GFP expression occurs preferentially in cells that express Keratin13 mRNA and hardly at all in those that are deleted for the Keratin13 gene. This effect is due to the fact that in the absence of Keratin13 mRNA, the blocker sequence within the generated mRNA construct interacts with the IRES sequence and, by altering the IRES secondary structure, suppresses its function and thus GFP expression. In contrast, in the presence of Keratin13 mRNA (WT), the generated mRNA construct is degraded down to the loops by sense-antisense interaction, whereby the blocker sequence disappears. This releases the function of the IRES and results in GFP expression. 5.) Quantitative evaluation of the functional system for the selective expression of target genes ( Figure 16).

[0050] Wild type and Keratin13 mutant cells were prepared as in Fig. 4 The cells were loaded with the different functional systems as described above. After 24 hours, the expression level of the selected target protein GFP was measured in all cells using flow cytometry. At least 5000 cells were analyzed for each measurement. All differences between WT and Keratin13- / - mutant were highly significant.

Claims

1. Method for targeted translation of a polypeptide of interest in a eukaryotic target cell, characterized by the introduction of an RNA construct into the target cell, the RNA construct having at least the following elements in the 5' to 3' direction: (a) an anticodogenic element (a) that is complementary to at least a portion of an mRNA of a gene expressed by the target cell; (b) a blocking element (b) that interacts with at least a portion of the translation initiator element (d) at the 3' position; (c) a stabilizing element (c); (d) a translation initiator element (d) that has an internal ribosomal entry site (IRES) sequence; (e) a sequence (e) encoding the polypeptide of interest, the translation of the polypeptide of interest being prevented in a eukaryotic non-target cell by base pairing of the blocking element (b) with the translation initiator element (d), and the translation of the encoded polypeptide taking place in a eukaryotic target cell due to processing of the RNA construct induced by the interaction of the anticodogenic element with the mRNA expressed by the target cell.

2. Method according to claim 1, wherein the interaction of the anticodogenic element (a) with the mRNA expressed by the target cell results in degradation or inactivation of the blocking element (b), so that translation of the polypeptide of interest can take place.

3. Method according to claim 2, wherein the degradation of the mRNA is stopped at the stabilizing element (c).

4. Method according to one or more of claims 1 to 3, characterized in that the anticodogenic element (a) has one or more of the following features: (i) attachment to an RNA sequence specific to the target cell. (ii) formation of double-stranded RNA hybrids of freely adjustable length (iii) induction of cellular degradation of the introduced RNA construct in the region of the double-stranded RNA hybrid.

5. Method according to one or more of claims 1 to 4, characterized in that the blocking element (b) has one or more of the following features: (i) the function of the translation initiator element (d) is suppressed. (ii) the blocking element (b) can be degraded or inactivated under suitable conditions. (iii) the suppressive function on the translation initiator element (d) is reversible after inactivation or degradation of the blocking element. (iv) the blocking element (b) performs its function by base pairing in or near the sequence of the translation initiator element (d) and thereby alters the secondary structure thereof. (v) the blocking element (b) performs its function by causing other molecules to directly or indirectly inhibit the translation initiator element (d).

6. Method according to one or more of claims 1 to 5, characterized in that the stabilizing element (c) has one or more of the following features: (i) nucleotide sequence that prevents degradation of RNA by exo- and / or endonucleases in the 3' direction. (ii) nucleotide sequence, characterized in that it forms stabilizing secondary structures, such as stem loops. (iii) nucleotide sequence, characterized in that stabilizing elements such as peptides or proteins bind to the RNA. (iv) nucleotide sequence, characterized in that molecules bind to the RNA and perform a chemical, stabilizing modification of the RNA 5' end.

7. Method according to one or more of claims 1 to 6, characterized in that the translation initiator element (d) has one or more of the following features: (i) cell type-dependent, selective inhibitability of the translation initiation function (ii) functionality only upon degradation or inactivation of the components of the regulatory cassette (elements a and b) (iii) functionality exclusively in target cells, while the translation initiator element is inactive in all other cell types. (iv) induction of translation independent of the cap structure in the 5' region of the initial RNA construct. (v) induction of translation by direct or indirect binding of ribosomal subunits or complete ribosomes. (vi) induction of translation by formation of specific secondary structures after cessation of inhibition by the blocking element (vii) induction of translation by attachment of specific molecules or chemical modification after cessation of inhibition by the blocking element8. Method according to one or more of claims 1 to 7, characterized in that the RNA construct has one or more of the following optional features: (i) 5' cap sequence in order to increase the stability of the entire mRNA construct (ii) 5' untranslated region in order to be able to regulate the stability of the entire mRNA construct and to suppress the 5' cap induced translation (e.g. incorporation of quadruplex sequences) (iii) 3' untranslated region in order to regulate the stability of the entire mRNA construct as well as the processed mRNA fragment in target cells. (iv) poly(A) region in order to regulate the stability of the entire mRNA construct as well as the processed mRNA fragment in target cells. (v) all elements of the entire mRNA construct from claims 1 to 8 may contain linker sequences of any length within the element itself or between the individual elements. Linker sequences can be functionless and used purely to spatially separate the individual elements, or they can integrate additional functions into the system.

9. RNA construct for targeted translation of a polypeptide of interest in a eukaryotic target cell, wherein the RNA construct has at least the following elements in the 5' to 3' direction: (a) an anticodogenic element (a) that is complementary to at least a portion of an RNA of a DNA portion expressed by the target cell, or induces target cell-specific degradation; (b) a blocking element (b) that is complementary to or otherwise inhibits at least a portion of the translation initiator element (d) at the 3' position; (c) a stabilizing element (c) that restricts the target cell-specific degradation of the RNA to a section thereof; (d) a translation initiator element (d) that functions independently of the 5' cap and is only active in target cells; wherein the translation initiator element (d) has an internal ribosomal entry site (IRES) sequence; (e) a sequence (e) encoding the polypeptide of interest; wherein the translation of the polypeptide of interest is prevented in a eukaryotic non-target cell by base pairing of the blocking element (b) with the translation initiator element (d), and the translation of the encoded polypeptide in a eukaryotic target cell due to an interaction of the anticodogenic element (a) with an RNA expressed by the target cell, a specific processing of the RNA construct takes place, whereby the translation initiator element is activated.

10. RNA construct according to claim 9, characterized by one or more of the following features: (i) the anticodogenic element (a) has one or more of the features of claim 4; (ii) the blocking element (b) has one or more of the features of claim 5; (iii) the stabilizing element (c) has one or more of the features of claim 6; (iv) the translation initiator element (d) has one or more of the features of claim 7; and / or (v) the mRNA construct has one or more of the features of claim 8.