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WO2025036992A1 - Conjugués d'arn - Google Patents

Conjugués d'arn Download PDF

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Publication number
WO2025036992A1
WO2025036992A1 PCT/EP2024/073042 EP2024073042W WO2025036992A1 WO 2025036992 A1 WO2025036992 A1 WO 2025036992A1 EP 2024073042 W EP2024073042 W EP 2024073042W WO 2025036992 A1 WO2025036992 A1 WO 2025036992A1
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WO
WIPO (PCT)
Prior art keywords
rna
conjugate
rna conjugate
peptide
protein
Prior art date
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Pending
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PCT/EP2024/073042
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WO2025036992A9 (fr
Inventor
Jörg Braun
Elena KHAZINA
Rainer Schwarz
Thomas Schlake
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Curevac SE
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Curevac SE
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Publication of WO2025036992A1 publication Critical patent/WO2025036992A1/fr
Publication of WO2025036992A9 publication Critical patent/WO2025036992A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • RNA molecules that encode proteins or peptides provide a number of potential advantages for various clinical applications.
  • RNA can be transfected into cell in vivo, in vitro, or ex vivo to induce expression of desired therapeutic or diagnostic proteins for preventing, treating or diagnosing disease.
  • the therapeutic potential of RNA is e.g. limited by stability and half-life of the RNA, the level of expression of the encoded protein, or the immunogenicity caused by the RNA molecule.
  • an object of the invention is to provide RNA molecules that are inter alia improved in respect of expression, stability and half-life, or immunogenicity.
  • the present invention is inter alia directed to an RNA conjugate that comprises at least one element A and at least one element B, wherein element A comprises or consists of an RNA molecule comprising at least one coding sequence encoding at least one peptide or protein, and wherein element B comprises or consists of at least one element C (sealing element).
  • element B may be conjugated to element A via a linker element L.
  • the RNA conjugate is advantageously characterized by e.g. an increased resistance to degradation in a cell and/or a prolonged expression of the encoded peptide or protein in a cell. Further aspects inter alia relate to methods of producing the RNA conjugates, methods for increasing the expression of an RNA, and medical uses of the RNA conjugate, composition, or kit.
  • RNA conjugates of the invention can inter alia increase and/or prolong the production of an encoded protein in a cell and/or reduce the induction of inflammatory cytokines.
  • the present invention relates to an RNA conjugate comprising at least one element A and at least one element B, wherein element A comprises or consists of an RNA molecule comprising at least one coding sequence encoding at least one peptide or protein, and wherein element B preferably comprises or consists of at least one element C (at least one sealing element).
  • element B is conjugated to element A via a linker element L.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the RNA conjugate of the first aspect.
  • the RNA conjugate is formulated in lipid-based carriers, for example lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • the present invention relates to a combination comprising at least one RNA conjugate and at least one different RNA molecule, e.g. an mRNA.
  • the present invention relates to a kit or kit of parts comprising at least one RNA conjugate of the first aspect, at least one pharmaceutical composition of the second aspect, or at least one combination of the third aspect.
  • the present invention relates to the RNA conjugate of the first aspect, the pharmaceutical composition of the second aspect, the combination of the third aspect, or the kit or kit of parts of the fourth aspect for use as a medicament.
  • the present invention relates to methods of treating or preventing a disease, disorder, or condition comprising applying or administering to a subject in need thereof the RNA conjugate of the first aspect, the pharmaceutical composition of the second aspect, the combination of the third aspect, and/or the kit or kit of parts of the fourth aspect.
  • RNA conjugate of the invention and a method of increasing or prolonging protein expression of an RNA molecule.
  • Artificial RNA refers to a nucleic acid that does not occur naturally.
  • an artificial RNA may be understood as a non-natural RNA molecule.
  • Such RNA molecules may be non-natural due to their individual sequence (e.g. G/C content modified cds, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides.
  • artificial RNA may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides.
  • an artificial RNA is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type sequencerhe naturally occurring sequence by at least one nucleotide.
  • the term is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical RNA molecules.
  • Cationic, cationizable means that the respective structure bears a positive charge, either permanently or not permanently, for example in response to certain conditions such as pH.
  • cationic covers both “permanently cationic” and “cationisable”.
  • permanently cationic means, e.g., that the respective compound, or group, or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge results from the presence of a quaternary nitrogen atom.
  • cds The terms “coding sequence” and the corresponding abbreviation “cds” as used herein refers to a sequence of several nucleotide triplets, which may be translated into a peptide or protein.
  • a cds in the context of the present invention may be a DNA or RNA sequence consisting of a number of nucleotides that may be divided by three, which starts with a start codon, and which preferably terminates with a stop codon.
  • Derived from The term “derived from” as used herein in the context of a nucleic acid e. for a nucleic acid “derived from”
  • (another) nucleic acid means that the nucleic acid, which is derived from (another) nucleic acid, shares at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or are identical with the nucleic acid from which it is derived.
  • the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence, shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or are identical with the amino acid sequence from which it is derived.
  • fragment as used herein in the context of a nucleic acid sequence (e.g RNA or DNA) or an amino acid sequence may typically be a shorter portion of a reference sequence of e.g. a nucleic acid sequence or an amino acid sequence.
  • a fragment typically consists of a sequence that is identical to the corresponding stretch within the reference sequence.
  • a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total reference molecule from which the fragment is derived.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule .e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosine-monophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogs thereof, which are connected to each other along a so-called backbone.
  • the backbone is typically formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • RNA sequence The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence.
  • RNA can be obtained by transcription of a DNA sequence, e.g., inside a cell or in vitro. In the context of the invention, the RNA may be obtained by RNA in vitro transcription or chemical synthesis.
  • Variant refers to a variant of a nucleic acid sequence derived from another nucleic acid sequence.
  • a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
  • a variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from.
  • a variant may be a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
  • a “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% nucleotide identity over a stretch of at least 30, 50, 75 or 100 nucleotides.
  • variant in the context of proteins or peptides refers to a protein or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s)/substitution(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants Preferably, these fragments and/or variants have the same, or a comparable specific property. Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three- dimensional structure by insertion® or deletion® can easily be determined e.g. using CD spectra (circular dichroism spectra).
  • a variant of a protein may be a functional variant of the protein, which means that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the function of the protein it is derived from.
  • a “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 30, 50, 75 or 100 amino acids of such protein or peptide.
  • the invention provides an RNA conjugate.
  • the RNA conjugate is characterized by an increased or prolonged half-life, an increased resistance to degradation, an increased or prolonged stability, an increased expression (of the encoded protein), a reduced induction of innate immune response (e.g. reduced induction of proinflammatory cytokines) and/or an increased translation efficiency when introduced into a population of cells.
  • the RNA conjugate comprises at least one “element B” that comprises or consists of at least one “element C” or “sealing element” as further specified herein. Suitable features and embodiments relating to element B are provided herein (see section “Detailed description of element B”).
  • a “sealing element” in the context of the invention refers to an element for conjugation to an RNA molecule to obtain an RNA conjugate of the invention.
  • a sealing element confers a certain functionality to the RNA molecule.
  • a sealing element is located at the 3’ end of the RNA molecule and therefore “seals” the tail of the RNA molecule.
  • the terms “sealing element” and “element C” have to be used interchangeably in the context of the invention.
  • the RNA conjugate comprises at least one “element A” that comprises or consists of at least one RNA molecule.
  • the RNA molecule comprises at least one cds encoding at least one peptide or protein. Suitable features and embodiments relating to element A are provided herein (see section “Detailed description of element A).
  • the RNA conjugate comprises at least one element A and at least one element B, wherein element A comprises or consists of an RNA molecule comprising at least one cds encoding at least one peptide or protein, and wherein element B comprises or consists of at least one element C (at least one sealing element).
  • the at least one element A as defined herein and the at least one element B as defined herein are conjugated to form an RNA conjugate.
  • element B is conjugated to the 3'-terminus of element A. Accordingly, in preferred embodiments, the RNA conjugate comprises a 5’ terminal element A followed by element B, wherein element B is conjugated to element A.
  • the RNA conjugate is an artificial RNA that does not occur in nature.
  • element A has been conjugated to element B by stepwise solid-phase conjugation, synthetic conjugation or post-synthetic conjugation.
  • the post-synthetic conjugation comprises a solid phase conjugation or in solution conjugation.
  • the post synthetic conjugation is an in-solution conjugation.
  • element B has been conjugated to element A, the RNA molecule, via reductive amination of the periodate-oxidized RNA.
  • element B is conjugated to element A via a linker element L.
  • Suitable features and embodiments relating to element L are provided herein (see paragraph “Detailed description of element L”).
  • the RNA conjugate comprises the elements A, L, and B, wherein element A comprises or consists of an RNA molecule comprising at least one cds encoding at least one peptide or protein, wherein element B comprises or consists of at least one element C (at least one sealing element), and wherein element B is conjugated to element A via a linker element L.
  • the RNA conjugate comprises a 5’ terminal element A followed by a linker element L followed by element B, wherein element B is conjugated to element A via a linker element L.
  • element A has been linked to element B by annealing methods, e.g. in embodiments where element B comprises a nucleic acid sequence.
  • element A has been conjugated to element B by ligation methods known in the art.
  • element A has been conjugated to element B by click chemistry.
  • the RNA conjugate comprises at least one element B that comprises or consists of at least one element C I sealing element (throughout the invention, “element C” and “sealing element” are used interchangeably).
  • element B comprises at least two sealing elements (at least two element C) as defined herein, for example 2, 3, 4, or even more sealing elements.
  • the sealing element is selected from at least one peptide, at least one protein, at least one nucleoside, at least one nucleotide, at least one nucleic acid (e.g., oligonucleotide or polynucleotide), at least one targeting moiety, at least one small molecule, or at least one polymer.
  • the sealing element is selected from at least one peptide, at least one protein, at least one nucleoside, at least one nucleotide, at least one nucleic acid (e.g., oligonucleotide or polynucleotide), at least one targeting moiety, at least one small molecule, or at least one polymer.
  • the sealing element (the element C) comprises a modified sealing element selected from at least one modified peptide, at least one modified protein, at least one modified nucleoside, at least one modified nucleotide, at least one modified nucleic acid (e.g., oligonucleotide or polynucleotide), at least one modified targeting moiety, at least one modified small molecule, at least one modified peptide, at least one modified protein, or at least one modified polymer.
  • a modified sealing element selected from at least one modified peptide, at least one modified protein, at least one modified nucleoside, at least one modified nucleotide, at least one modified nucleic acid (e.g., oligonucleotide or polynucleotide), at least one modified targeting moiety, at least one modified small molecule, at least one modified peptide, at least one modified protein, or at least one modified polymer.
  • modified sealing element relates to modifications that may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo compared to sealing elements without the modification.
  • the term “modified sealing element” relates to modifications comprising elements or structures to enable the conjugation to element A.
  • the term “modified sealing element” relates to modifications comprising elements or structures to enable the conjugation to element A via a linker, preferably a linker as specified herein.
  • modified sealing elements relates to modifications comprising elements to improve the conjugation reaction in time or efficiency to build the RNA conjugate of the invention.
  • the sealing element (elements C) used for generating the RNA conjugate comprises structures, bonds, or elements to enable the conjugation to element A preferably via forming element L.
  • the sealing element (the element C) used for generating the RNA conjugate comprises a nucleophile group for conjugating it to element A preferably via forming element L.
  • the sealing element used for generating the RNA conjugate is a nucleophile comprising a nucleophile group.
  • nucleophile is an atom or molecule that has an affinity for atomic nuclei or that donates electrons to form a bond, preferably a covalent bond.
  • Nucleophilic groups are those which have electron-rich atoms able to donate a pair of electrons to form a new covalent bond.
  • the most relevant nucleophilic atoms are oxygen, nitrogen, and sulfur, and the most common nucleophilic functional groups are water, alcohols, phenols, amines, thiols, and occasionally carboxylates, preferably amines.
  • the nucleophile group of the sealing element (the element C) used for generating the RNA conjugate is an amine group or activated amine group.
  • a preferred “amine group” in the context of the invention is an amino group (-NH2).
  • the “activated amine group” in the context of the invention comprises a hydrazine, a hydrazide, a hydrazinonicotinic acid or an oxyamine.
  • the sealing element in particular the sealing element the (element C) used for generating the RNA conjugate, comprises element NH2-(M)n- to provide the amine group or activated amine group for conjugation.
  • the sealing element in particular the sealing element (the element C) used for generating the RNA conjugate, comprises the element NH2-(M)n- to enable conjugation to element A preferably via forming element L (see e.g. reaction scheme 2 of Example 1 .3).
  • the sealing element (the element C), in particular the sealing element used for generating the RNA conjugate, comprises element NH2-(M)n-, wherein (M)n comprises O, NR N , a bond, optionally substituted C1 -C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2- C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted diethylen glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C
  • the sealing element (element C) comprises NH2-(M)n-, wherein (M)n comprises a bond, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, substituted C5 alkyl or C6 alkyl, and wherein n is 1 .
  • SEQ ID sequences disclosed herein which describe sequences of element B may become modified during the conjugation of element B to element A.
  • a SEQ ID NO providing the sequence of element B may carry a terminal -NH2 that, during the process of RNA conjugate formation, may become modified while forming linker element L.
  • the resulting RNA conjugate, comprising element B may lack said terminal -NH2 of element B because the N of the -NH2 is then part of linker element L.
  • element B comprises at least one sealing element (element C) selected from at least one peptide.
  • the RNA conjugate comprises at least one peptide as defined herein.
  • RNA conjugates comprising at least one peptide are characterized by an increased translation (of the encoded peptide protein) and/or a prolonged translation (of the encoded peptide protein) when administered to a cell.
  • the sealing element comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 peptides.
  • the at least one peptide comprises about 1 to about 50 amino acids, about 10 to about 50 amino acids, preferably about 15 to 20 amino acids. In a specific embodiment, the peptide comprises 17 amino acids.
  • the at least one peptide comprises at least one modified amino acid.
  • the modified amino acid comprises at least one modification, preferably a chemical or post-translational modification such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, and/or a combination of these, or synthetic or non-natural amino acids or D-amino acids.
  • a chemical or post-translational modification such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, and/or a combination of these, or synthetic or non-natural amino acids or D-amino acids.
  • the at least one peptide is selected from or derived from a coactivator in the regulation of translation initiation, a peptide regulating mRNA degradation and stability, a nuclear localization peptide, an ER localization peptide, an enhancer of an antigen-presentation peptide, an endosomal escape peptide, an immune stimulation peptide, a Golgi apparatus localization peptide, a lysosomal localization peptide, a mitochondrial localization peptide, and/or a peptide for purification or affinity chromatography, or a fragment or variant of any of these.
  • the at least one peptide is selected from or derived from a nuclear localization peptide that has an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to any one of SEQ ID NO: 1-16 of patent application WO2022232945.
  • the at least one peptide is selected from or derived from a peptide that influences the stability and/or translational efficiency of the RNA molecule.
  • the at least one peptide has an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequence PFVYLI of patent application WD2021094792.
  • the at least one peptide is selected from or derived from a coactivator in the regulation of translation initiation.
  • coactivator in the regulation of translation initiation relates to a stimulatory activity on translation mediated via binding or recruiting or enhancing of translation initiation factors (e.g. elF1 , elF2, elF3, elF4, elF4A, elF4G, elF4E, elF5, elF6, PABP).
  • translation initiation factors e.g. elF1 , elF2, elF3, elF4, elF4A, elF4G, elF4E, elF5, elF6, PABP.
  • the at least one peptide is selected from or derived from a coactivator in the regulation of translation initiation, selected from Polyadenylate-binding protein-interacting protein, preferably Polyadenylate-binding protein-interacting protein 1 and/or Polyadenylate-binding protein-interacting protein 2 (PABP-interacting protein 1 or 2; PAIP-1 or -2).
  • Polyadenylate-binding protein-interacting protein acts as a coactivator in the regulation of translation initiation of polypcontaining mRNAs. Its stimulatory activity on translation is mediated via its action on PABPC1 . Its association with elF4A and PABPC1 potentiate contacts between mRNA termini.
  • the at least one peptide of the Polyadenylate-binding protein-interacting protein 1 and/or Polyadenylate-binding protein-interacting protein 2 comprises or consists of an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to any one of SEQ ID NOs: 1-8, ora fragment or variant of any of these sequences.
  • the at least one peptide is selected from or derived from a PABPC1 -interacting motif of the Polyadenylate-binding protein-interacting protein.
  • the at least one peptide is selected from or derived from a PABPC1 -interacting motif 1 or 2 of the Polyadenylate-binding protein-interacting protein 1 or 2.
  • the at least one peptide comprises at least one amino acid sequence selected or derived from a PABPC1 -interacting motif of a Polyadenylate-binding protein-interacting protein (PAIP) being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1 or 2, or fragments or variants of any of these.
  • PAIP Polyadenylate-binding protein-interacting protein
  • the at least one peptide is selected from or derived from a PABPC1 -interacting motif 2 of the Polyadenylate-binding protein-interacting protein 1 .
  • the at least one peptide of the PABPC1 -interacting motif 2 comprises or consists of an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to SEQ ID NO: 1 , or a fragment or variant of any of these sequences.
  • the at least one peptide is selected from or derived from a PABPC1 -interacting motif 2 of the Polyadenylate-binding protein-interacting protein 2.
  • the at least one peptide of the PABPC1 -interacting motif 2 comprises or consists of an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to SEQ ID NO: 2, or a fragment or variant of any of these sequences.
  • coactivator in the regulation of translation initiation relates to a stimulatory activity on translation mediated via binding or recruiting or enhancing of translation initiation factors (e.g. elF1 , elF2, elF3, elF4, elF4A, elF4G, elF4E, elF5, elF6, PABP).
  • translation initiation factors e.g. elF1 , elF2, elF3, elF4, elF4A, elF4G, elF4E, elF5, elF6, PABP.
  • the at least one peptide is selected from or derived from elF4G.
  • a peptide comprises a PABP RRM2-interacting motif.
  • the at least one peptide of elF4G comprises or consists of an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to any one of SEQ ID NOs: 145, ora fragment or variant of any ofthese sequences.
  • the N-terminus of the at least one peptide is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the C-terminus of the at least one peptide is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the N-terminus of the at least one peptide is conjugated to the 3’ terminus of element A.
  • the at least one peptide used for generating the RNA conjugate comprises a nucleophile group. In embodiments, the at least one peptide used for generating the RNA conjugate comprises a nucleophile group, preferably an amine group. In some embodiments, the at least one peptide used for generating the RNA conjugate comprises an element NH2-(M)n- to enable conjugation to the RNA molecule, preferably by forming the linker of formula I. In embodiments, the at least one peptide used for generating the RNA conjugate comprises an element NH2-(M)n- at the N-terminus.
  • the at least one peptide is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least one sealing element (element C) selected from at least one nucleoside or nucleotide.
  • element C sealing element selected from at least one nucleoside or nucleotide.
  • the RNA conjugate comprises at least one nucleoside or nucleotide as defined in the following.
  • the at least one nucleoside or nucleotide comprises an adenine, guanine, cytosine, thymine or uracil.
  • the nucleoside or nucleotide is a modified nucleoside or modified nucleotide.
  • the sealing element comprises one modified nucleoside or nucleotide.
  • the sealing element includes a plurality of modified nucleotides.
  • the sealing element comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleotides.
  • Modified nucleosides and nucleotides have useful properties including increased stability and/orthe lack of a substantial induction of the innate immune response, e.g. induction of proinflammatory cytokines, of a cell into which the RNA conjugate comprising the modified nucleoside or modified nucleotide is introduced.
  • the at least one modified nucleoside or nucleotide is selected from, or comprises a modified nucleobase selected from, modification list 1 as disclosed in paragraph “Chemical modifications” in section “Detailed Description of element A - RNA molecule”.
  • the modified nucleoside and/or modified nucleotide is selected from the group of L-adenosine, 2’-0-methyl-adenosine, phosphorothioat-2-O-methyl-adenosine, alpha-thio-2-O-methyl-adenosine, N6-methyl- adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine, PNA-adenosine, or inverted thymidine.
  • the modification of the at least one nucleoside and/or nucleotide is selected from the group of phosphorothioate, phosphorodithioate, methyl phosphonate, mesyl phosphoramidate, boranophosphate, LNA (locked nucleic acid), 2’-0-methylation (of ribose), 2’-Methoxyethoxy (of ribose), Methoxyethyl (of ribose), phosphordiamidate morpholino oligomer, 2 -O-methyl-phosphorothioate, 2’-deoxy-2’-a-fluoro, deoxynucleotide, 2’-0-Methoxymethyl (of ribose), phosphorodiamidate morpholino oligonucleotides, 2-O-Propargyl, L-Ribose, dihydro-base, inverted base, hexitol-base, arabino-su
  • the sealing element includes a chain termination nucleoside or nucleotide such as 3’- deoxyadenosine (cordycepin), 3’-deoxyuridine, 3’-deoxycytidine, 3’-deoxyguanosine, 3’-deoxythymidine, 2’, 3’- dideoxynucleoside, 2’,3’-dideoxyadenosine, 2’,3’-dideoxyuridine, 2’,3’-dideoxycytidine, 2’,3’-dideoxyguanosine, 2’, 3’- dideoxythymidine, 2’-deoxynucleoside, or an O-methylnucleoside.
  • a chain termination nucleoside or nucleotide such as 3’- deoxyadenosine (cordycepin), 3’-deoxyuridine, 3’-deoxycytidine, 3’-deoxyguanosine, 3’-deoxythymidine, 2’
  • the sealing element includes G-quadroduplexsuch as G4.
  • G is referring to a Guanin (DNA, RNA, LNA, or PNA)
  • N is referring to a Linker of up to 7 nucleotides (DNA, RNA, LNA, or PNA).
  • the sealing element comprises at least two different modified nucleosides, preferably at least one modified nucleoside is 2’-0-methyl-adenosine and at least one modified nucleoside is inverted thymidine. In some embodiments the modified nucleoside is not an inverted thymidine.
  • the 5’ end of the at least one nucleotide or nucleoside is conjugated to the 5’ terminus or 3 terminus of element A or non-terminal to element A.
  • the 3’ end of the at least one nucleotide or nucleoside is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the 3‘end of the at least one nucleotide or nucleoside is conjugated to the 3’ terminus of element A.
  • the at least one (modified) nucleotide or (modified) nucleoside used for generating the RNA conjugate comprises a nucleophile group. In embodiments, the at least one (modified) nucleoside or (modified) nucleotide used for generating the RNA conjugate comprises element NH2-(M)n- to conjugate to element A. In embodiments, the at least one (modified) nucleoside is conjugated to the RNA molecule via a linker, preferably a linker of formula I.
  • the at least one (modified) nucleoside is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least one sealing element (element C) selected from at least one nucleic acid.
  • the RNA conjugate comprises at least one nucleic acid as a sealing element (element C) as defined in the following.
  • the at least one nucleic acid of element B does not comprise a cds.
  • the at least one nucleic acid of element B has not been prepared by RNA in vitro transcription.
  • the at least one nucleic acid of element B has been prepared by chemical synthesis.
  • the at least one nucleic acid may comprise dNTPs, NTPs, LNAs, PNAs, and/or modified dNTPs, NTPs, LNAs, PNAs, or any combination thereof.
  • the at least one nucleic acid has a length of about 2 to about 500 nucleotides, preferably about 2 to about 100 nucleotides.
  • the nucleic acid of element B is a linear nucleic acid or a branched nucleic acid.
  • the at least one nucleic acid is selected from an oligonucleotide (that typically comprises about 2 to about 25 nucleotides) or from a polynucleotide (that typically comprises more than 25 nucleotides, for example 30 to 500 nucleotides or 30 to 100 nucleotides).
  • the at least one nucleic acid comprises at least one modified nucleotide or modification.
  • Modified nucleotides have useful properties including increased stability and/orthe lack of a substantial induction of the innate immune response of a cell into which the oligonucleotides or polynucleotides comprising the modified nucleotide are introduced.
  • the at least one nucleic acid comprises at least one modified nucleotide or modification selected from the group of phosphorothioate, phosphorodithioate, methyl phosphonate, mesyl phosphoramidate, boranophosphate, LNA (locked nucleic acid), 2’-Q-methylation (of ribose), 2’-Methoxyethoxy (of ribose), Methoxyethyl (of ribose), phosphordiamidate morpholino oligomer, 2-O-methyl-phosphorothioate, 2’-deoxy-2’-a-fluoro, deoxynucleotide, 2-0- Methoxymethyl (of ribose), phosphorodiamidate morpholino oligonucleotides, 2 -O-Propargyl, L-Ribose, dihydro-base, inverted base, hexitol-base, arabino-sugar
  • the at least one modified nucleotide or modification is selected from the modification phosphorothioate, 2’- O-methylation (of ribose), 2-O-methyl-phosphorothioate and/or PNA (peptide nucleic acid).
  • the at least one nucleic acid comprises at least one modified adenosine, preferably selected from L-adenosine, 2’-0-methyl-adenosine, alpha-thio-2-O-methyl-adenosine, phosphorothioat-2-O- methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine or PNA-adenosine.
  • modified adenosine preferably selected from L-adenosine, 2’-0-methyl-adenosine, alpha-thio-2-O-methyl-adenosine, phosphorothioat-2-O- methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine or PNA-a
  • the at least one modified nucleotide of the nucleic acid comprises at least one PNA modification, preferably a PNA-adenosine.
  • the at least one modified adenosine is selected from PNA-adenosine comprising the structure of formula II
  • the nucleic acid comprises modified adenosines, preferably PNA-adenosines comprising the structure of formula II.
  • the at least one nucleic acid comprises a plurality of adenosines, preferably modified adenosines, wherein the plurality of adenosines comprises about 4 to about 100 adenosines, preferably 4 to 60 adenosines, more preferably 4 to 40 adenosines.
  • the plurality of adenosines is a consecutive sequence of adenosines, preferably modified adenosines.
  • the nucleic acid that comprises a plurality of adenosines is branched, e.g. a branched poly(A)sequence.
  • the branched poly(A) may comprise at least 2, 3, or more nucleic acid sequences that comprise or consists of a poly(A)sequence.
  • the nucleic acid comprises 4 or 6 modified adenosines, preferably PNA-adenosines comprising the structure of formula II, wherein q is 3 or 5.
  • the nucleic acid comprises 4 modified adenosines, preferably PNA-adenosines comprising the structure of formula II, wherein q is 3.
  • the nucleic acid comprises 6 modified adenosines, preferably PNA-adenosines comprising the structure of formula II, wherein q is 5.
  • the nucleic acid comprises 10 modified adenosines, preferably PNA-adenosines comprising the structure of formula II, wherein q is 9.
  • the nucleic acid comprises or consists of (PNA)-A4, (PNA)-A6, (PNA)-A10 (SEQ ID NO: 13).
  • the nucleic acid comprises or consists of (PNA)-A6-hydrazine (NH2NH-CH2-CO-AAAAAA).
  • the at least one modification of the modified nucleotide is selected from 2’-Q-methylation, preferably a 2’-O-methylated guanosine or2’-Q-methylated adenosine.
  • the modification is selected from 2-O-methyl-phosphorothioate or phosphorothioate, preferably 2’-O-methyl phosphorothioate.
  • the modified nucleotide is a modified adenosine, preferably 2-O-methyl- phosphorothioate adenosine.
  • the nucleic acid comprises 16 modified adenosines, preferably 162-O-methyl- phosphorothioate adenosines. In particularly preferred embodiments, the nucleic acid comprises 32 modified adenosines, preferably 32 phosphorothioate adenosines. In particularly preferred embodiments, the nucleic acid comprises 32 modified adenosines, preferably 322-O-methyl-phosphorothioate adenosines.
  • the RNA conjugate comprises a nucleic acid being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 14, 15, 16 or 17, or a fragment or variant thereof.
  • the at least one nucleic acid comprises an aptamer, a riboswitch, a purification handle, a locked nucleic acid, a miRNA binding site or a PABP-affinity sequence.
  • the at least one nucleic acid comprises at least one antagonist of at least one RNA sensing pattern recognition receptor.
  • RNA sensing pattern recognition receptor antagonist efficiently antagonizes the immunostimulation of RNA, an unwanted side-effect that is typically triggered by RNA sensing receptors.
  • the RNA sensing pattern recognition receptor antagonist is a Toll-like receptor antagonist, preferably a TLR7 antagonist and/or a TLR8 antagonist. In some embodiments, the RNA sensing pattern recognition receptor antagonist comprises at least one modified nucleotide.
  • the at least one modified nucleotide of the RNA sensing pattern recognition receptor antagonist is selected from the group of modified guanosine, modified adenosine or modified uridine.
  • the modified guanosine of the RNA sensing pattern recognition receptor antagonist is a 2’-0-methyl-guanosine (indicated herein as Gm orG(2’OMe)) or phosphorothioate guanosine (indicated herein as G*) or2’-G-methyl-phosphorothioate-guanosine (indicated herein as G(2’OMe)*).
  • the modified adenosine of the RNA sensing pattern recognition receptor antagonist is a 2’-O-methyl- adenosine (indicated herein A(2’OMe)) or phosphorothioate adenosine (indicated herein as A*) or2’-0-methyl- phosphorothioate-adenosine (indicated herein as A(2’OMe)*).
  • the modified uridine of the RNA sensing pattern recognition receptor antagonist is a 2’-O-methyl-uridine (indicated herein U(2’OMe)) or phosphorothioate uridine (indicated herein as U*) or2’-0-methyl-phosphorothioate-uridine (indicated herein as U(2’OMe)*).
  • the modified cytidine of the RNA sensing pattern recognition receptor antagonist is a 2’-O-methyl-cytidine (indicated herein C(2’OMe)) or phosphorothioate cytidine (indicated herein as C*) or2’-0-methyl- phosphorothioate-cytidine (indicated herein as C(2’OMe)*).
  • the RNA sensing pattern recognition receptor antagonist comprises or consists of at least one nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 85-212 of WD2021028439, or fragments or variants of any of these.
  • the at least one TLR7/8 antagonist comprises a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence GAGCGmGCCA, or SEQ ID NO: 149, ora fragment ora variant of any ofthese.
  • the at least one TLR7/8 antagonist comprises a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence SEQ ID NO: 18 according to sequence *A(2’OMe)*U(2’OMe)*A*A(2’OMe)*U(2’OMe)*U*U*U*U(2’OMe) *U(2’OMe)*G*G*U*A(2’OMe)*U(2’OMe)*U*U or a fragment or a variant of any ofthese.
  • the at least one nucleic acid comprises at least 4 nucleotides, preferably 4 guanosines.
  • the at least one nucleic acid is conjugated to the 5’ or 3’ terminus of element A or non-terminal to element A. In preferred embodiments, the at least one nucleic acid is conjugated to the 3’ terminus of element A.
  • the at least one nucleic acid used for generating the RNA conjugate comprises at least one nucleophile group.
  • the nucleophile group of the nucleic acid used for generating the RNA conjugate is an amine group or an activated amine group.
  • the at least one nucleic acid used for generating the RNA conjugate comprises element NH2-(M)n-, preferably at the 5’ terminus of the nucleic acid.
  • the at least one nucleic acid used for generating the RNA conjugate comprises an element NH2-(M)n- to enable conjugation to the RNA molecule, preferably by forming the linker of formula I.
  • the at least one nucleic acid used for generating the RNA conjugate comprises NH2-(M)n, wherein (M)n is further specified in M is a bond, C6 alkyl, C2 alkyl, substituted C1 alkyl or substituted C5 alkyl, and n is 1 .
  • the at least one nucleic acid is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • the at least one nucleic acid of element B is annealed to the RNA molecule, e.g. via a stretch of complementary nucleotides (e.g. a stretch of at least 5, 6, 7, 8 or more nucleotides).
  • the at least one nucleic acid of element B comprises a cds.
  • the at least one nucleic acid of element B has been prepared by RNA in vitro transcription.
  • the at least one nucleic acid of element B may be a coding RNA such as an mRNA.
  • the coding RNA preferably the mRNA, is conjugated to the nucleic acid of element A via a 3’ to 3’ connection.
  • an RNA conjugate may comprise two mRNA molecules that are conjugated to each other via a 3’ to 3’ link.
  • a 3’ to 3’ link may be obtained by linker elements as defined herein, or, alternatively, by click chemistry.
  • such an RNA conjugate may have the following structure: 5’ cap - UTR - cds - UTR - Poly(A) - 3’-3’ Link - poly(A) - UTR - cds - UTR - 5’ cap.
  • element B comprises at least one sealing element (element C) selected from at least one small molecule.
  • the RNA conjugate comprises at least one small molecule as defined in the following.
  • small molecule refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory orfound in nature.
  • Small molecules can referto compounds that are “natural product-like”, however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it possesses one or more of the following characteristics including having several carboncarbon bonds, having multiple stereocenters, having multiple functional groups, having at least two different types of functional groups, and having a molecular weight of less than 1500 Da, preferably less than 1000 Da, although this characterization is not intended to be limiting for the purposes of the disclosure.
  • the at least one small molecule comprises a benzyl group (e.g., O-benzylhydroxylamine, O- (2,3,4,5,6-petafluorobenzyl)hydroxylamine, O-tritylhydroxylamine, O-(4-nitro-benzyl) hydroxylamine).
  • a benzyl group e.g., O-benzylhydroxylamine, O- (2,3,4,5,6-petafluorobenzyl)hydroxylamine, O-tritylhydroxylamine, O-(4-nitro-benzyl) hydroxylamine.
  • the small molecule comprises an alkyl group (e.g., methoxyamine, O-ethylhydroxylamine, O-tert- butylhydroxylamine, O-tert-butyldimethylsilylhydroxylamine, O-(carboxymethyl) hydroxylamine, Spermidine).
  • alkyl group e.g., methoxyamine, O-ethylhydroxylamine, O-tert- butylhydroxylamine, O-tert-butyldimethylsilylhydroxylamine, O-(carboxymethyl) hydroxylamine, Spermidine.
  • the at least one small molecule is selected from spermidine (formula VIII of Table 2).
  • the at least one small molecule comprises methoxyamine comprising the structure of formula VI (formula VI)
  • the at least one small molecule comprises a hydrazine group (e.g. methylhydrazine, 1 ,1- Dimethylhydrazine, phenylhydrazine, 2,4-Dinitrophenylhydrazine, 1 ,2-Diphenylhydrazine, Tetraphenylhydrazine), ora hydrazide ora hydrazinonicotinic acid.
  • a hydrazine group e.g. methylhydrazine, 1 ,1- Dimethylhydrazine, phenylhydrazine, 2,4-Dinitrophenylhydrazine, 1 ,2-Diphenylhydrazine, Tetraphenylhydrazine
  • a hydrazine group e.g. methylhydrazine, 1 ,1- Dimethylhydrazine, phenylhydrazine, 2,4-Dinitrophenylhydrazine, 1 ,2-Diphen
  • the at least one small molecule is methylhydrazine comprising the structure of formula V (formula V)
  • the at least one small molecule comprises a heterocycle or homocycle (e.g., O-(tetra-2H-pyran- 2-yl) hydroxylamine or 10-[2-(aminooxy)ethyl]-1 OH-phenothiazine, Naphthylamine, 1-Bicyclo[1.1.1]pentylamine).
  • a heterocycle or homocycle e.g., O-(tetra-2H-pyran- 2-yl) hydroxylamine or 10-[2-(aminooxy)ethyl]-1 OH-phenothiazine, Naphthylamine, 1-Bicyclo[1.1.1]pentylamine.
  • the at least one small molecule comprises Naphthylamine or 1-Bicyclo[1 .1 ,1]pentylamine.
  • the at least one small molecule comprises a heteroatom (e.g., hydroxylamine-O-sulfonic acid or hydroxylamine).
  • a heteroatom e.g., hydroxylamine-O-sulfonic acid or hydroxylamine
  • the at least one small molecule comprises a bile acid (e.g. cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glycoursodeoxycholic acid (GUDCA)).
  • CA cholic acid
  • DCA deoxycholic acid
  • DCA deoxycholic acid
  • LCA glycocholic acid
  • TCA glycodeoxycholic acid
  • GDCA
  • the at least one small molecule comprises a sugar (e.g. ribose, D-ribose, L-ribose, a-D- ribopyranose, p-D-ribopyranose, p-D-ribofuranose, a-D-ribofuranose, arabinose, xylose, lyxose, deoxyribose).
  • a sugar e.g. ribose, D-ribose, L-ribose, a-D- ribopyranose, p-D-ribopyranose, p-D-ribofuranose, a-D-ribofuranose, arabinose, xylose, lyxose, deoxyribose.
  • the at least one small molecule comprises D-Glucosamine, preferably comprising the structure of formula IV1 or IV2. (formula IV2)
  • the at least one small molecule comprises an amino acid, preferably amino acids with a L, D, S or R convention (e.g. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine).
  • a L, D, S or R convention e.g. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.
  • the at least one small molecule is Lysine (comprising the structure of formula XI) or L-Tryptophan (comprising the structure of formula VII) (formula VII)
  • the at least one small molecule is selected from the group of Methylhydrazine, Methoxyamine, D-Glucosamine, Lysine, L-Tryptophan, spermidine, 1-Bicyclo[1.1.1]pentylamine hydrochlorid (formula IX of Table 2) and/or 1 -Naphthylamine (formula X of Table 2).
  • the at least one small molecule is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the at least one small molecule is conjugated to the 3’ terminus of element A.
  • the at least one small molecule used to generate the RNA conjugate comprises a nucleophile group, preferably an amine group. In embodiments, the at least one small molecule used to generate the RNA conjugate comprises element NH2-(M)n- to conjugate to element A.
  • the at least one small molecule is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least one sealing element (element C) selected from at least one protein.
  • the RNA conjugate comprises at least one protein as defined herein.
  • the sealing element comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins.
  • the protein is at least 50 amino acids in length. In one embodiment, the protein has a length ranging from 50 amino acids to 1000 amino acids.
  • the at least one protein comprises at least one modified amino acid.
  • the modified amino acid comprises at least one modification selected from chemical or post- translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, and/or a combination of these, or synthetic or non-natural amino acids.
  • chemical or post- translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, and/or a combination of these, or synthetic or non-natural amino acids.
  • the at least one protein is selected from or derived from a coactivator in the regulation of translation initiation and/or control, nuclear localization proteins, ER localization proteins, endosomal escape proteins, post translation modification proteins, ribosome binding proteins, immune stimulation proteins, Golgi apparatus localization proteins, lysosomal localization proteins, mitochondrial localization proteins, and/or proteins for purification or affinity chromatography, or fragments or variant of any of these.
  • the at least one protein influences the stability and/or translational efficiency of the RNA conjugate.
  • the at least one protein is modified according to the following features selected from modified in transcriptional and translational control regions; inserted or removed protein trafficking sequences; removed/added post translation modification sites (e.g., glycosylation sites); added, removed or shuffled protein domains; inserted or deleted restriction sites; modified ribosome binding sites and mRNA degradation sites; modified to adjust translational rates; modified to allow the various domains of the protein to fold properly; or modified to reduce or eliminate secondary structures with the RNA molecule.
  • the at least one protein is a lipoprotein.
  • the N-terminus of the at least one protein is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the C-terminus of the at least one protein is conjugated to the 5’ terminus or 3’ terminus of element A or non-terminal to element A.
  • the N-terminus of the at least one protein is conjugated to the 3’ terminus of element A.
  • the at least one protein used to generate the RNA conjugate comprises a nucleophile group, preferably an amine. In some embodiments, the at least one protein used to generate the RNA conjugate comprises element NH2- (M)n- to conjugate to element A.
  • the at least one protein is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least one sealing element (element C) selected from at least one targeting moiety.
  • the RNA conjugate comprises at least one targeting moiety as defined in the following.
  • the at least one targeting moiety can bind to a biological target either covalently or non-covalently.
  • biological targets include biopolymers, e.g., antibodies, nucleic acids such as RNA and DNA, proteins, aptamers, enzymes; exemplary proteins include enzymes, receptors, and ion channels.
  • the target is a tissue- or cell-type specific marker, e.g., a protein that is expressed specifically on a selected tissue or cell type.
  • the biological target is a receptor, such as, but not limited to, plasma membrane receptors and nuclear receptors; more specific examples include G-protein-coupled receptors, cell pore proteins, transporter proteins, surface-expressed antibodies, HLA proteins, MHC proteins and growth factor receptors.
  • a receptor such as, but not limited to, plasma membrane receptors and nuclear receptors; more specific examples include G-protein-coupled receptors, cell pore proteins, transporter proteins, surface-expressed antibodies, HLA proteins, MHC proteins and growth factor receptors.
  • the at least one targeting moiety comprises a cell penetrating moiety or agent that enhances intracellular delivery of the RNA conjugate of the invention.
  • the at least one targeting moiety comprises a cell-penetrating peptide sequence that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cellpenetrating peptides and/or combinations of any thereof.
  • a cell-penetrating peptide sequence that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cellpenetrating peptides and/or combinations of any thereof.
  • the at least one targeting moiety comprises a carbohydrate, a lipid, a vitamin, a small receptor ligand, a cell surface carbohydrate binding protein or a ligand thereof, a lectin, an r-type lectin, a galectin, a ligand to a duster of differentiation (CD) antigen, CD30, CD40, a cytokine, a chemokine, a colony stimulating factor, an interferon, a interleukin, a lymphokine, a monokine, ora mutant, derivative and/or combinations of any thereof.
  • CD duster of differentiation
  • the at least one targeting moiety comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 different or identical targeting moieties.
  • the at least one targeting moiety is conjugated to the 3’ terminus or 5’ terminus of element A or non-terminal to element A.
  • the at least one targeting moiety is conjugated to the 3’ terminus of element A.
  • the at least one targeting moiety used for generating the RNA conjugate comprises a nucleophile group.
  • the nucleophile group of the targeting moiety used for generating the RNA conjugate is an amine group or an activated amine group.
  • the at least one targeting moiety used for generating the RNA conjugate comprises element NH2-(M)n- to conjugate to element A.
  • the at least one targeting moiety is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least one sealing element (element C) selected from at least one polymer.
  • element C comprises at least one sealing element selected from at least one polymer.
  • the RNA conjugate comprises at least one polymer as defined in the following.
  • the at least one polymer comprises therapeutic agents, detectable substances, contrast agents, polyethylene glycol (PEG), polypropylene glycol, a cationic polymer, or any synthetic or naturally occurring macromolecule made up of repeating monomeric units.
  • the polymer comprises a therapeutic agent such as a cytotoxin, a radioactive ion, a chemotherapeutic, or another therapeutic agent.
  • a therapeutic agent such as a cytotoxin, a radioactive ion, a chemotherapeutic, or another therapeutic agent.
  • the polymer is a therapeutic agent such as a cytotoxin, a radioactive ion, a chemotherapeutic, or another therapeutic agent.
  • Suitable cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol, CC-1065 and analogs or homologs thereof.
  • Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, Samarium 153 and praseodymium.
  • therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC- 1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mit
  • the polymer is a detectable substance.
  • Suitable detectable substances include various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent materials, radioactive materials, and contrast agents.
  • Suitable optically-detectable labels include for example, without limitation, 4-acetamido-4’-isothiocyanatostilbene-2,2- disulfonic acid; acridine and derivatives; acridine, acridine isothiocyanate; 5-(2-aminoethyl)aminonaphthalene-1 -sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-naphthyl)maleimide; anthranilamide; BODI PY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4’,6-diaminidino
  • Cyanine-5 (Cy5); Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
  • the detectable label is a fluorescent dye, such as Cy5 and Cy3.
  • the detectable agent is a non-detectable precursorthat becomes detectable upon activation.
  • examples include fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, ortetrazine-BODI PY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSE (VisEn Medical)).
  • the polymer is a luminescent material.
  • luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin.
  • the polymer comprises a radioactive material.
  • Suitable radioactive materials include 18F, 67Ga, 81 mKr, 82Rb, 1111n, 1231, 133Xe, 201TI, 1251, 35S, 14C, 3H, or 99mTc (e.g. as pertechnetate (technetate (VI I), Tc04-) either directly or indirectly, or other radioisotope detectable by direct counting of radio emission or by scintillation counting.
  • the polymer is a contrast agent.
  • Suitable contrast agents include contrast agents for MRI or NMR, for X-ray CT, Raman imaging, optical coherence tomography, absorption imaging, ultrasound imaging, orthermal imaging can be used, e.g. gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g. superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons can also be used.
  • gold e.g., gold nanoparticles
  • gadolinium e.g., chelated Gd
  • iron oxides e.g. superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and
  • the polymer is a polyethylene glycol (PEG), polypropylene glycol, a cationic polymer, or any synthetic or naturally occurring macromolecule made up of repeating monomeric units.
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • a cationic polymer or any synthetic or naturally occurring macromolecule made up of repeating monomeric units.
  • the polymer is a polyethylene glycol (PEG) or a cationic polymer.
  • the polymer is an optionally substituted straight chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer. In some embodiments, the polymer is an optionally substituted branched chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer.
  • the polymer is an optionally substituted branched polysaccharide or an optionally substituted unbranched polysaccharide.
  • the polymer is an optionally substituted polyethylene glycol, polypropylene glycol, or polyvinyl alcohol or derivative thereof.
  • the polymer is a branched chain polyethylene glycol, polypropylene glycol, or polyvinyl alcohol or derivative thereof.
  • the polymer is polyethylene glycol (PEG) or a derivatized form of PEG (e.g., N- hydroxylsuccinimide (NHS) active esters of PEG such as succinimidyl propionate, benzotriazole active esters, and PEG derivatized with maleimide, vinyl sulfones, or thiol groups).
  • PEG polyethylene glycol
  • NHS N- hydroxylsuccinimide
  • the at least one polymer is conjugated to the 5’ terminus or the 3’ terminus of element A or nonterminal to element A. In preferred embodiments, the at least one polymer is conjugated to the 3’ terminus of element A.
  • the at least one polymer used to generate the RNA conjugate comprises a nucleophile group. In embodiments, the at least one polymer used to generate the RNA conjugate comprises element NH2-(M)n- to conjugate to element A.
  • the at least one polymer is conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • element B comprises at least two sealing elements (elements C), preferably selected from peptide, protein, nucleoside, nucleotide, nucleic acid, targeting moiety, small molecule, and/or polymer.
  • the RNA conjugate comprises at least two sealing elements as defined herein.
  • the at least two sealing elements (elements C) are selected from a nucleic acid as defined herein, preferably an oligonucleotide, and a nucleoside or nucleotide as defined herein.
  • the at least two sealing elements are selected from a peptide, preferably a NLS peptide, and a small molecule, preferably a cholic acid.
  • the at least two sealing elements are conjugated to the 5’ terminus orthe 3’ terminus of element A or non-terminal to element A. In preferred embodiments, the at least two sealing elements are conjugated to the 3’ terminus of element A.
  • the at least two sealing elements are conjugated to the RNA molecule via a linker element, preferably a linker element L as defined herein (e.g. according to formula I).
  • the RNA conjugate comprises at least one element A that comprises or consists of at least one RNA molecule.
  • the RNA molecule may be any type of RNA including any type of single stranded RNA, double stranded RNA, and/or linear RNA.
  • the RNA molecule is selected from an mRNA, a replicon RNA, a self-replicating RNA, or viral RNA.
  • the RNA molecule is an mRNA.
  • the RNA conjugate comprises at least one mRNA as the element A and at least one element B comprising at least one sealing element (element C) as defined herein.
  • the RNA molecule comprises about 50 to about 20000 nucleotides, about 500 to about 10000 nucleotides, about 1000 to about 10000 nucleotides, preferably about 1000 to about 5000 nucleotides, or even more preferably about 2000 to about 5000 nucleotides.
  • the RNA molecule comprises a 5’-cap structure.
  • Such a 5’-cap structure suitably stabilizes the RNA molecule and/or enhances expression of the encoded protein and/or reduces the stimulation of the innate immune system after administration.
  • the RNA molecule comprises a 5’-cap structure, preferably m7G, capO, cap1 , cap2, a modified capO ora modified cap1 structure.
  • 5’-cap structure as used herein will be recognized and understood by the person of ordinary skill in the art and is e.g. intended to referto a 5’ modified nucleotide, particularly a guanine nucleotide, positioned at the 5’-end of an RNA, e.g. an mRNA.
  • the 5’-cap structure is connected via a 5’-5’-triphosphate linkage to the RNA.
  • a 5’-cap (capO or cap1) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co- transcriptional capping) using cap analogues.
  • cap analogue refers to a non-polymerizable di-nucleotide or tri-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of an RNA molecule when incorporated at the 5’-end of the RNA molecule.
  • Non-polymerizable means that the cap analogue will be incorporated only at the 5’-terminus because it does not have a 5’ triphosphate and therefore cannot be extended in the 3’-direction by a templatedependent polymerase, particularly, by template-dependent RNA polymerase.
  • a cap1 or modified cap1 structure is generated using a cap analog, preferably a tri-nucleotide cap analog.
  • a cap analog preferably a tri-nucleotide cap analog.
  • Any cap analog derivable from the structures defined in claims 1-13 of WO2017053297 (hereby incorporated by reference) or, alternatively, any cap analog derivable from the structures defined in claim 1-37 ofW02023007019 (hereby incorporated by reference) may be suitably used to co-transcriptionally generate a cap1 or modified cap1 .
  • the 5’-cap structure may suitably be added co-transcriptionally using tri-nucleotide cap analogue as defined herein, preferably in an RNA in vitro transcription reaction as defined herein.
  • the RNA molecule comprises a cap1 structure ora modified cap1 structure.
  • the cap1 structure is formed via co-transcriptional capping using tri-nucleotide cap analogues m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG.
  • the cap1 structure is formed using co-transcriptional capping using tri-nucleotide cap analogue 3’-O-Me-m7G(5’)ppp(5’)G, m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG, 3’OMe-m7G(5’)ppp(5’)(2’OMeA)pG or 3’OMe-m7G(5’ppp(5’)(2’OMeG)pG, particularly preferred 3'OMe-m7G(5’)ppp(5’)(2’OMeA)pG.
  • the RNA molecule comprises modified capO, cap1 or cap2 structures that protect the oxidation of the 5’ terminus of the RNA molecule during the conjugation step of element A to B.
  • the RNA molecule comprises modified cap 0, cap1 or cap2 structures that protect the oxidation of the 5’ terminus of the RNA molecule to allow a defined conjugation step to the 3’ terminus of element A during post-synthetic conjugation.
  • the modified cap structure is a cap analogue without any diol group.
  • the RNA molecule comprises a 5’ terminus with the following structure: 3’OMe- m7G(5’)ppp(5')(2’OMeA)pG.
  • the cap1 structure is a modified cap1 structure and is formed using co- transcriptional capping using tri-nucleotide cap analogue as defined and described in W02023007019, preferably in Figure 1 (e.g. based on the m7Guanine-diethyleneglycole-pppN cap structure).
  • the 5’-cap structure is formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or cap-dependent 2-0 methyltransferases) to generate capO or cap1 or cap2 structures.
  • capping enzymes e.g. vaccinia virus capping enzymes and/or cap-dependent 2-0 methyltransferases
  • RNA comprises a cap structure, preferably a cap1 structure as determined by a capping assay (e.g. via an assay as described in cl. 27 to 46 of W02015101416).
  • the RNA comprises a 5’-terminal sequence element comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequences GGGAGA, AGGAGA, GGGAAA, AGAAUA, AGAUUA, GAUGGG or GGGCG, or a fragment or variant of these sequences, preferably AGGAGA, ora fragment or variant.
  • a 5’-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase.
  • the first nucleotide of said 5’-terminal start sequence may preferably comprise a 2’0 methylation, e.g. 2’0 methylated guanosine ora 2’0 methylated adenosine.
  • 5’UTR/3’UTR 5’UTR/3’UTR:
  • the RNA molecule comprises at least one untranslated region (UTR).
  • UTR untranslated region
  • the at least one untranslated region is selected from at least one 5-UTR and/or at least one 3-UTR.
  • UTR untranslated region
  • UTR element refers to a part of an RNA molecule typically located 5’ or 3’ of a coding sequence.
  • An UTR is not translated into protein.
  • An UTR may be part of the RNA molecule.
  • An UTR may comprise elements for controlling gene expression, also called regulatory elements.
  • regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements, translation stop elements etc.
  • the RNA molecule comprises at least one cds encoding at least one peptide or protein, and a 5-UTR and/or 3-UTR.
  • UTRs may harbour regulatory sequence elements that determine RNA turnover, stability, and localization.
  • UTRs may harbour sequence elements that enhance translation.
  • translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy.
  • Certain combinations of 3’-UTRs and/or 5’-UTRs may enhance the expression of operably linked coding sequences encoding peptides or proteins as defined herein.
  • RNA molecules harbouring said UTR combinations advantageously enable rapid and transient expression of encoded protein or peptide after administration to a subject.
  • the RNA molecule comprises at least one heterologous 5-UTR and/or at least one heterologous 3-UTR.
  • Said heterologous 5’-UTRs or3’-UTRs may be derived from naturally occurring genes or may be synthetically engineered.
  • the RNA molecule comprises at least one cds as defined herein operably linked to at least one (heterologous) 3-UTR and/or (heterologous) 5-UTR.
  • the RNA comprises at least one 5 -UTR, preferably at least one heterologous 5-UTR.
  • 5’-untranslated region refers to a part of an RNA located 5’ (i.e. “upstream”) of a cds and which is not translated into protein.
  • a 5-UTR may be part of an RNA located 5’ of the cds.
  • a 5-UTR starts with the transcriptional start site and ends before the start codon of the cds.
  • a 5 -UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, aptamers etc.
  • the RNA molecule comprises at least one 5-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
  • the 5-UTR is synthetic.
  • the RNA molecule comprises at least one 5-UTR, wherein the at least one 5-UTR comprises a nucleic acid sequence derived or selected from a 5 -UTR of gene selected from HSD17B4, AIG1 , alphaglobin (HBA1 , HBA2), ASAH1 , ATP5A1 , COX6C, DPYSL2, HHV5, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL32, RPL35A, SLC7A3, synthetic origin, TUBB4B, UBQLN2, or from a homolog, a fragment or variant of any one of these genes.
  • the at least one 5-UTR derived or selected from HSD17B4, AIG1 , alpha-globin (HBA1 , HBA2), ASAH1 , ATP5A1 , COX6C, DPYSL2, HHV5, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL32, RPL35A, SLC7A3, synthetic origin, TUBB4B, UBQLN2, comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 30-83, ora fragment ora variant of any of these.
  • the RNA molecule comprises a 5 -UTR derived or selected from a HSD17B4 gene.
  • the at least one 5-UTR comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 31 or 83, ora fragment ora variant of any of these.
  • the RNA molecule comprising at least one cds encoding at least one peptide or protein comprises at least one 3’-UTR, preferably at least one heterologous 3’-UTR.
  • 3’-untranslated region refers to a part of an RNA molecule located 3’ (i.e. downstream) of a cds and which is not translated into protein.
  • a 3’-UTR may be part of a nucleic acid located between a cds and an (optional) terminal poly(A) sequence.
  • a 3’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.
  • the RNA molecule comprises at least one 3’-UTR, which may be derivable from a gene that relates to an RNA with enhanced half-life (i.e. that provides a stable RNA).
  • the 3’-UTR is synthetic.
  • the 3’-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.
  • the 3’-UTR comprises at least one translation stop element in the 5’ end of the 3’UTR.
  • the RNA molecule comprises at least one 3’-UTR, wherein the at least one 3’-UTR comprises or consists of a nucleic acid sequence derived or selected from a 3’-UTR of a gene selected from PSMB3, AES-12S, ALB7, alpha-globin (HBA1 , HBA2), ANXA4, beta-globin (HBB), CASP1 , COX6B1 , FIG4, GH1 , GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B, or from a homolog, a fragment, or variant of any one of these genes.
  • a gene selected from PSMB3, AES-12S, ALB7, alpha-globin (HBA1 , HBA2), ANXA4, beta-globin (HBB), CASP1 , COX6B1 , FIG4, GH1 , GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B, or from a homo
  • the at least one 3’-UTR that is derived or selected from PSMB3, AES-12S, ALB7, alphaglobin (HBA1 , HBA2), ANXA4, beta-globin (HBB), CASP1 , COX6B1 , FIG4, GH1 , GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 84-141 , or a fragment ora variant of any of these.
  • the RNA molecule comprises a 3’-UTR derived or selected from a PSMB3 gene.
  • the at least one 3-UTR comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 85, 131, 133, 135, 137, 139 or 141 , more preferably SEQ ID NO: 85, or a fragment or a variant of any of these.
  • the RNA molecule comprises at least one cds encoding at least one peptide or protein as defied herein and is operably linked to a 3’-UTR and/or a 5’-UTR selected from the 5’-UTR/3’-UTR combinations (5’UTR/3’UTR) provided in WG2021239880 [p.127, line 35 to p.128, line 2], which is hereby incorporated by reference.
  • the at least one 5’-UTR is selected from HSD17B4 and the at least one 3’-UTR is selected from PSMB3.
  • the RNA molecule is monocistronic, bicistronic, or multicistronic, preferably monocistronic.
  • the RNA molecule comprises a ribosome binding site, also referred to as “Kozak sequence” identical to or at least 80%, 85%, 90%, 95% identical to any one of SEQ ID NOs: 143, 144, or sequences GCCGCCACC (DNA), GCCGCCACC (RNA), GCCACC (DNA), GCCACC (RNA), ACC (DNA) or ACC (RNA), or fragments or variants of any of these.
  • the “Kozak sequence” comprises or consists of RNA sequence ACC.
  • the RNA molecule comprises a 5’-terminal sequence element comprising or consisting of an RNA sequence, being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequences GGGAGA, AGGAGA, GGGAAA, AGAAUA, AGAUUA, GAUGGG or GGGCG, or a fragment or variant of these sequences, preferably AGGAGA, ora fragment or variant.
  • a 5’-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase.
  • the first nucleotide of said 5’-terminal start sequence may preferably comprise a 2’0 methylation, e.g. 2’0 methylated guanosine ora 2’0 methylated adenosine.
  • the RNA molecule is modified, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.
  • a modified RNA molecule may comprise nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications.
  • a backbone modification in the context of the invention is a modification in which phosphates of the backbone of the nucleotides of the RNA molecule are chemically modified.
  • a sugar modification in the context of the invention is a chemical modification of the sugar of the nucleotides of the RNA molecule.
  • a base modification in the context of the invention is a chemical modification of the base moiety of the nucleotides of the RNA molecule.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable fortranscription and/or translation.
  • the RNA molecule comprises at least one modified nucleotide or nucleoside.
  • the at least one modified nucleotide or nucleoside is selected from, or comprises a modified nucleobase selected from, the following modification list 1 : a modified uracil or uridine including pseudouridine (qi), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5- bromo-uracil), 3-methyl-uracil (m3U), 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, 99% or 100% of the nucleotides in the RNA molecule are replaced with modified nucleotides.
  • essentially all, e.g. essentially 100% of the nucleotides in the coding sequence or the full RNA sequence have a chemical modification, preferably a chemical modification in the 5-position of the uracil.
  • the at least one modified nucleotide is N1 -methylpseudouridine (m1 qi).
  • 100% of the uracils in the RNA molecule is substituted with N1 -methylpseudouridine (ml qi).
  • the RNA molecule does not comprise chemically modified nucleotides.
  • a 5’-cap structure as defined below is typically not considered to be a chemically modified nucleotide. Accordingly, in that context, the RNA molecule consists only of G, C, A and U nucleotides and therefore does not comprise modified nucleotides, and optionally comprises a 5’-cap structure. Accordingly, in embodiments, the RNA molecule does not comprise N1- methylpseudouridine (ml 1 ) substituted positions or pseudouridine (qi) substituted positions.
  • the RNA molecule comprises at least one cds encoding at least one peptide or protein.
  • the RNA molecule comprising a cds encoding 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins or peptides.
  • the RNA molecule comprises at least one cds encoding at least one peptide or protein suitable for use in treatment or prevention of a disease, disorder or condition.
  • the at least one peptide or protein is selected or derived from an antibody, an intrabody, a receptor, a receptor agonist, a receptor antagonist, a binding protein, a CRISPR-associated endonuclease, a chimeric antigen receptor (CAR), a chaperone, a transporter protein, an ion channel, a membrane protein, a secreted protein, a transcription factor, a toxin, an enzyme, a peptide or protein hormone, a growth factor, a structural protein, a cytoplasmic protein, a cytoskeletal protein, an allergen, a tumor antigen, a proto-oncogene, an oncogene, a tumor suppressor gene, a neoantigen, a mutated antigen, an antigen of a pathogen, or any fragment, epitope, variant, or combination thereof.
  • CAR chimeric antigen receptor
  • the RNA molecule is a modified and/or stabilized nucleic acid.
  • the RNA molecule may thus be provided as a “stabilized RNA” that is to say a nucleic acid showing improved resistance to in vivo degradation and/or a nucleic acid showing improved stability in vivo, and/or a nucleic acid showing improved translatability in vivo.
  • the at least one cds of the RNA molecule is a codon modified cds.
  • the amino acid sequence encoded by the at least one codon modified cds is not being modified compared to the amino acid sequence encoded by the corresponding wild type or reference cds.
  • codon modified cds relates to a cds that differs in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference cds.
  • a codon modified cds may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably to optimize/modify the cds for in vivo applications.
  • the at least one cds is a codon modified cds, wherein the codon modified cds is selected from C maximized cds (as further defined in WC2021239880 [p.122, lines 33 to 39] which is hereby incorporated by reference); a CAI maximized cds (as further defined in WC2021239880 [p.123, lines 33 to 44] which is hereby incorporated by reference); a human codon usage adapted cds (as further defined in WC2021239880 [p.123, lines 7 to 17] which is hereby incorporated by reference); a G/C content modified cds (as further defined in WC2021239880 [p.123, lines 19 to 31] which is hereby incorporated by reference); and G/C optimized cds (“opt1”), or any combination thereof.
  • the at least one codon modified cds is a G/C optimized cds.
  • the nucleic acid may be modified, wherein the G/C content of the at least one cds may be optimized compared to the G/C content of the corresponding wild type or reference cds (herein referred to as “G/C optimized cds”).
  • G/C optimized cds compared to the G/C content of the corresponding wild type or reference cds.
  • “Optimized” in that context refers to a cds wherein the G/C content is preferably increased to the essentially highest possible G/C content.
  • the generation of a G/C content optimized nucleic acid sequence may be carried out using a method according to W02002098443. In this context, the disclosure of W02002098443 is included in its full scope in the present invention.
  • G/C optimized cds are indicated by the abbreviations “opt1”.
  • the at least one cds has a G/C content of at least about 50%, 55%, or 60%. In particular embodiments, the at least one cds has a G/C content of at least about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
  • the at least one cds comprises more than one stop codon to allow sufficient termination of translation. In particularly embodiments, the at least one cds comprises two or three stop codons to allow sufficient termination of translation. These more than one stop codons may optionally be positioned in alternative reading frames.
  • the RNA molecule comprises at least one poly(N) sequence, preferably at least one poly(A) sequence. In some embodiments the RNA molecule does not comprise a poly(A) sequence.
  • the RNA molecule comprises at least two, three, or more poly(A) sequences.
  • poly(A) sequence refers to be a sequence of adenosine nucleotides, typically located at the 3’-end of a linear RNA of upto about 1000 adenosine nucleotides. Typically, said poly(A) sequence is homopolymeric. Alternatively, a poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide.
  • the at least one poly(A) sequence comprises about 20 to about 500 adenosines, about 40 to about 250 adenosines, about 60 to about 250 adenosines, preferably about 60 to about 150 adenosines. In embodiments, the at least one poly(A) sequence comprises about or more than 10, 30, 50, 64, 75, 100, 200, 300, 400, or 500 adenosines.
  • the length of the poly(A) sequence comprises less than about 30 consecutive adenosine nucleotides, for example 10 to 30 consecutive adenosine nucleotides.
  • the at least one poly(A) sequence comprises about 64 adenosine nucleotides (A64), preferably about 64 consecutive adenosine nucleotides. In particularly preferred embodiments, the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.
  • the RNA molecule comprises at least one interrupted poly(A) sequence, wherein the poly(A) sequence is interrupted by at least one non-adenosine nucleotide (N), preferably by about 10 non-adenosine (N10) nucleotides.
  • N non-adenosine nucleotide
  • N non-adenosine nucleotide
  • a poly(A) sequence A30-N10-A70 is preferred.
  • the poly(A) sequence as defined herein is located directly at the 3’ terminus of the RNA molecule (element A). Accordingly, the 3’-terminal nucleotide (that is the last 3’-terminal nucleotide in the polynucleotide chain) is the 3’-terminal A nucleotide of the at least one poly(A) sequence.
  • the term “directly located at the 3’ terminus” has to be understood as being located exactly at the 3’ terminus of element A (e.g. A100 or A30
  • the RNA molecule comprises a poly(A) sequence of about 100 consecutive adenosine nucleotides, wherein said poly(A) sequence is located directly at the 3’ terminus of the RNA molecule, optionally wherein the 3’ terminal nucleotide is an adenosine
  • the poly(A) sequence of the RNA molecule is obtained from a DNA template during RNA in vitro transcription.
  • the poly(A) sequence is obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template.
  • poly(A) sequences are generated by enzymatic polyadenylation, wherein the majority of RNA molecules preferably comprise about 100 (+/- 20) to about 500 (+/- 100) adenosine nucleotides, preferably about 100 (+/- 20) to about 200 (+/- 40) adenosine nucleotides.
  • the RNA molecule comprises at least one polyadenylation signal.
  • the RNA molecule comprises 4 guanosines in the 3’ region of the Poly(A) sequence.
  • the RNA molecule comprises at least one poly(C) sequence and/or histone stem-loop sequence.
  • the RNA molecule comprises at least one histone stem-loop (hSL) or histone stem loop structure.
  • the term refers to nucleic acid sequences that forms a stem-loop secondary structure predominantly found in histone mRNAs.
  • a hSL in the context of the invention may be located in an UTR region, preferably in the 3’-UTR region.
  • a hSL may be derived from formulae (I) or (II) of WD2012019780.
  • the RNA molecule comprises at least one hSL sequence derived from at least one of the specific formulae (la) or (Ila) of W02012019780.
  • the histone stem-loop sequence comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 19 or 20, or a fragment or variant of any of these.
  • the histone stem-loop sequence comprises or consists of a nucleic acid sequence according to SEQ ID NO: 20, or a fragment or thereof.
  • the RNA molecule comprises a 3’-terminal sequence element.
  • the 3’-terminal sequence element represents the 3’ terminus of the RNA molecule of element A.
  • a 3’-terminal sequence element may comprise at least one poly(A) and/or Poly(C) sequence as defined herein and, optionally, at least one hSL as defined herein.
  • the RNA molecule comprises a 3’-terminal sequence element comprising a hSL as defined herein followed by a poly(A) sequence comprising about 100 consecutive adenosines. In other embodiments, the RNA molecule comprises a 3’-terminal sequence element comprising about 64 consecutive adenosines.
  • the RNA molecule comprises at least one 3’-terminal sequence element comprising or consisting of an RNA sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 21-29, ora fragment or variant of these sequences, preferably SEQ ID NOs: 21 or 22, or a fragment or variant thereof.
  • the RNA molecule of element A comprises the following elements, preferably in the following order (5’ to 3’ direction):
  • the RNA molecule of element A is preferably an in vitro transcribed RNA (e.g. an in vitro transcribed mRNA). Accordingly, the RNA molecule of the RNA conjugate is produced by RNA in vitro transcription.
  • RNA in vitro transcription relates to a process wherein RNA is synthesized in a cell-free system in vitro.
  • the RNA is obtained by transcribing a DNA template in the presence of a DNA-dependent RNA polymerase (e.g. 17, SP6), ribonucleotide triphosphates (NTPs, and optionally modified NTPs) and optionally, a cap analog, in an appropriate buffer (e.g. comprising MgCI 2 ).
  • a DNA-dependent RNA polymerase e.g. 17, SP6
  • NTPs ribonucleotide triphosphates
  • cap analog optionally, a cap analog
  • the nucleotide mixture for RNA in vitro transcription comprises modified nucleotides as defined herein.
  • preferred modified nucleotides may be selected from pseudouridine (qi) or N1- methylpseudouridine (m1 qi).
  • uracil nucleotides in the nucleotide mixture are replaced (either partially or completely) by pseudouridine (qi) and/or N1 -methylpseudouridine (ml qi) to obtain a modified RNA.
  • the nucleotide mixture used in RNA in vitro transcription does not comprise modified nucleotides as defined herein.
  • the nucleotide mixture used for RNA in vitro transcription does only comprise G, C, A and U nucleotides, and, optionally, a cap analog as defined herein to obtain a non-modified RNA (e.g. a non-modified mRNA).
  • the RNA molecule has been purified by at least one step of purification.
  • purified RNA refers to RNA which has a higher purity after certain purification steps than the starting material. Typical impurities comprise peptides, proteins, spermidine, RNA fragments, dsRNA, free nucleotides, DNA, etc. It is desirable for the “degree of RNA purity” to be as close as possible to 100%. Preferably, a “purified RNA” has a degree of purity of more than 75%, 80%, 85%, 90%, or 95%. The degree of purity may be determined by an analytical HPLC.
  • the RNA molecule has been purified by (RP)HPLC, AEX, size exclusion chromatography, hydroxyapatite chromatography, tangential flow filtration (IFF), filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, and/or cellulose-based purification.
  • the RNA has been purified by RP- HPLC (preferably as described in WD2008077592) and/or TFF (preferably as described in WD2016193206) and/or oligo d(T) purification (preferably as described in WD2016180430) e.g. to remove dsRNA, and/or RNA fragments.
  • the RNA molecule of element A has an integrity of at least 60%, 70%, 80%, 90%.
  • RNA integrity describes whether the complete RNA sequence is present. RNA integrity can be determined by RP-HPLC and may be based on determining the area under the peak of the expected full-length RNA in a chromatogram.
  • the purified RNA molecule is suitable for use in production of the RNA conjugate of the invention, preferably to conjugate element B to the RNA molecule of element A.
  • element B as defined herein is conjugated to element A as defined herein via a linker element L as further defined below.
  • the linker comprises a bond or linkage that conjugates element L directly to element A or B.
  • the linker comprises at least one non phosphodiester bond and the at least one non phosphodiester bond of element L is preferably conjugated directly to element A.
  • element B is conjugated covalently or non-covalently to element A via element L.
  • element B is conjugated covalently or non-covalently to the 3'-terminus of element A via element L, wherein a covalent conjugation is preferred.
  • element B becomes conjugated to element A via element L by stepwise solid-phase conjugation, synthetic conjugation, or post-synthetic conjugation, preferably post synthetic conjugation, to generate the RNA conjugate of the invention.
  • the stepwise solid phase conjugation is an on-line solid-phase synthesis or in-line solid-phase synthesis.
  • the element L of the stepwise solid phase conjugation is a bifunctional or trifunctional linker.
  • the post-synthetic conjugation comprises a solid phase conjugation or in solution conjugation, preferably in solution conjugation.
  • the conjugation of element B to element A via element L is a post synthetic in solution conjugation.
  • a solution in that context comprises two or more substances dissolved in a liquid form.
  • element B that has been used to generate the RNA conjugate comprises a nucleophile group for conjugating to element A via element L, preferably in a post synthetic in solution conjugation.
  • element B used for generating the RNA conjugate comprises a nucleophile group for reacting with an oxidized element of the 5’ terminus or 3’ terminus of element A, preferably the 3’ terminus of element A.
  • element L comprises a thioether or disulfide bond, a secondary amine, a tertiary amine, an oxime linkage, a thiazolidine linkage, a hydrazone linkage, an amide bond, a click chemistry linkage or/and a Diels-Alder reaction linkage.
  • linker element L comprises a tertiary amine or an amide bond.
  • element B used for generating the RNA conjugate comprises an amine group that during the process of conjugation reacts with an oxidized element ofthe 5’ terminus or 3’ terminus of element A, preferably the 3’ terminus of element A, more preferably with the 3’ terminus ofthe RNA molecule.
  • the RNA conjugate is a result of reductive amination of a periodate-oxidized RNA molecule.
  • element B used for generating the RNA conjugate comprises the element NH2-(M)n- that comprises a nucleophile group, wherein M is O, NR N , a bond, optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted diethylen glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C
  • element NH2-(M)n- of element B used for generating the RNA conjugate is reacting with an oxidized element of the 5’ terminus or 3’ terminus of element A, preferably of the 3’ terminus of element A, to generate the RNA conjugate of the invention.
  • the 3’ terminus of the RNA molecule of element A is a 3’ UTR, HSL, poly(A) sequence or poly(C) sequence. In preferred embodiments, the 3’ terminus of the RNA molecule of element A is a poly(A) sequence.
  • linker element L comprises the structure of formula I
  • G is a nucleobase, hydrogen, halo, hydroxy, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2- C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted amino, azido, optionally substituted C3-C10 (hetero)cycloalkyl, optionally substituted C6-C10 (hetero)aryl;
  • X is O, S, N(R3), or C(R3)2, wherein each R3 is, independently, H, halo, or optionally substituted C1-C10 alkyl;
  • R1 and R2 are each, independently, hydrogen, hydroxyl, or C1-C6 alkoxy, and wherein if G is nucleobase, R1 and R2 are hydrogen or hydroxyl;
  • M is O, NR N , a bond, optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted diethylen glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; n is O, 1 , 2, 3, 4, or 5;
  • A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position;
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase;
  • X is O;
  • R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen;
  • M is O, NR N , a bond, optionally substituted C1 -C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; n is 0 or
  • M is selected from a bond, C1 alkyl, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, C3 alkyl, substituted C3 alkyl, C4 alkyl, substituted C4 alkyl, C5 alkyl, substituted C5 alkyl, C6 alkyl, substituted C6 alkyl, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene; and n is selected from 0 or 1 .
  • M is selected from a bond, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, substituted C5 alkyl orC6 alkyl and n is 1 .
  • n is selected from 0.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a bond, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, substituted C5 alkyl orC6 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; n is 0; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a bond, n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a C6 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a substituted C5 alkyl, preferably an hydrazine substituted C5 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element L comprises the structure of formula I and formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a C5 alkylene; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • element A has been conjugated to element B via a chemical reaction, building the linker element L comprising the structure of formula I to generate an RNA conjugate of the invention.
  • element A has been treated to enable a conjugation with at least one element B via element L comprising the formula I to generate an RNA conjugate of the invention.
  • element L comprising the structure of formula I is derived from the sugar moiety at the 5'- terminus of an inverted cap structure.
  • an RNA molecule comprises a nucleic acid sequence of element A including an inverted cap structure which has been treated with an oxidant (e.g., sodium periodate) resulting in oxidative ring opening of the sugarto two aldehydes, followed by condensation of the dialdehyde with a nucleophile group provided by element B, preferably an amine.
  • an oxidant e.g., sodium periodate
  • element L comprising the structure of formula I is derived from the sugar moiety of the 3’ terminus of the last nucleotide of element A.
  • an RNA molecule comprises at the 3terminus a nucleotide treated with an oxidant (e.g., sodium periodate) which results in oxidative ring opening of the sugarto two aldehydes (see reaction scheme 1 , Example 1 .3), followed by condensation of the dialdehyde with a nucleophile group provided by element B, preferably an amine (reaction scheme 2, step 1 , Example 1 .3).
  • an oxidant e.g., sodium periodate
  • the produced RNA conjugate is reduced.
  • the RNA conjugate is reduced with a mild reducing agent.
  • the term “mild reducing agent” is used for agents that reduce RNA without causing damage to the RNA molecule.
  • the reducing agent is sodium cyanoborohydride (Na[BH3(CN)].
  • the reduced RNA conjugate is more stable under in vitro or in vivo (physiological) conditions, preferably in vivo conditions.
  • the RNA conjugate preferably the reduced RNA conjugate, is characterized by at least one of the following features selected from increased or prolonged half-life, increased resistance to degradation, increased or prolonged stability, increased expression, reduced induction of innate immune response, reduced induction of proinflammatory cytokines and/or an increased translation efficiency when introduced into a population of cells.
  • oxidation and condensation of the RNA molecule of element A has been carried out simultaneously to generate the RNA conjugate.
  • the conjugation reaction of element A to element B to generate the RNA conjugate has been carried out for 0.5h, 1 h, 1 ,5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11 h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or20h, preferably for 12h.
  • element B used to generate the RNA conjugate comprises additional elements or structures to improve the conjugation of element B to element A via element L.
  • the term “improve the conjugation” is defined in following but not limiting examples: reduce the chemical reaction duration, improve the chemical reaction efficiency, optimize the pH, optimize the ion concentration and/or optimized the temperature.
  • the additional element or structure improves the conjugation of sealing element B to element A, independently of element B.
  • the additional element or structure improves the conjugation of element B to element A, specifically for sealing element selected from the group of at least one nucleoside, nucleotide, oligonucleotide, polynucleotide, targeting moiety, small molecule, peptide, protein, or polymer.
  • the amount used of element B is adapted specifically for the sealing element used for the conjugation of element B to element A via element L.
  • the at least one additional element or structure reduces the chemical reaction time to 0.5h, 1 h, 1 ,5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11 h or 12h, preferably to less than 0.5h, 1 h, 1 ,5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h.
  • element B that has been used to generate the RNA conjugate comprises at least one activated amine group to improve the conjugation of element B to element A via element L.
  • the at least one activated amine group present in element B that has been used to generate the RNA conjugate reduces the chemical reaction time to 0.5h, 1 h, 1 ,5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11 h or 12h, preferably to less than 0.5h, 1 h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5 h.
  • the conjugation of element A to element B via element L has been carried out using a chemical reduction reaction by adding hydrochloride.
  • the RNA conjugate is a purified RNA conjugate.
  • the RNA conjugate has been purified by at least one step of purification. Accordingly, after the conjugation step of element B to element A (preferably a purified RNA molecule), the resulting RNA conjugate has been purified by at least one step of purification.
  • RNA conjugate as used herein has to be understood as an RNA conjugate that has a higher purity after certain purification steps than the starting material (e.g. the RNA conjugate after the conjugation step of element A and element B).
  • Typical impurities that are essentially not present in a purified RNA conjugate comprise non-conjugated element B nucleophiles, non-conjugated RNA molecules of element A, chemical compounds according to the conjugation process (e.g. Nal0 4 or NaBH 3 CN), peptides or proteins (e.g. enzymes derived from DNA dependent RNA in vitro transcription, e.g.
  • RNA polymerases RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double stranded RNA (dsRNA)), free nucleotides (modified nucleotides, conventional NTPs, cap analogue), template DNA fragments, buffer components (HEPES, TRIS, MgCI 2 ) etc.
  • dsRNA short double stranded RNA
  • free nucleotides modified nucleotides, conventional NTPs, cap analogue
  • template DNA fragments buffer components (HEPES, TRIS, MgCI 2 ) etc.
  • HEPES buffer components
  • TRIS TRIS, MgCI 2
  • Other potential impurities that may be derived from e.g. upstream processes such as RNA in vitro transcription, chemical synthesis of element B (e.g. peptide synthesis) etc. Accordingly, it is desirable in this regard for the
  • purified RNA conjugate as used herein has a degree of purity of more than 75%, 80%, 85%, very particularly 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or more.
  • the degree of purity is e.g. determined by an analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of the peak for the target RNA conjugate and the total area of all peaks including the peaks representing the by-products.
  • the degree of purity is e.g. determined by an analytical agarose gel electrophoresis or capillary gel electrophoresis.
  • purification of the RNA conjugate comprises at least one step of purification selected from RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, affinity chromatography, cellulose-based purification, or any combination thereof.
  • the RNA conjugate has been purified using RP-HPLC (preferably as described in WC2008077592) and/or TFF (preferably as described in WO2016193206) and/or oligo d(T) purification (preferably as described in WC2016180430).
  • the RNA conjugate has been purified using RP-HPLC.
  • the RNA conjugate has been purified using an affinity chromatography or capturing method selective for element B.
  • the RNA conjugate has a purity level of at least about 70%, 75%, 80%, 85%, 90%, or 95%, preferably more than 95%.
  • the degree of purity is determined by an analytical HPLC method.
  • the RNA conjugate has a certain RNA integrity. In embodiments, the RNA conjugate has an RNA integrity ranging from about 40% to about 100%. In embodiments, the RNA conjugate has an integrity of about 50%, about 60%, about 70%, about 80%, or about 90%. RNA integrity is suitably determined using analytical HPLC, preferably analytical RP-HPLC. In preferred embodiments, the RNA conjugate has an integrity of at least about 50%, preferably of at least about 60%, more preferably of at least about 70%, most preferably of at least about 80% or about 90% or higher. Integrity is suitably determined using analytical HPLC, preferably analytical RP-HPLC.
  • the purified RNA conjugate is suitable for use in treatment or prevention of a disease, disorder or condition.
  • the invention provides an RNA conjugate, wherein the conjugation of element A to element B results in improved characteristics of the RNA conjugate when compared to element A lacking element B.
  • the invention provides an RNA conjugate, wherein the RNA molecule of element A comprises at least one coding sequence encoding at least one peptide or protein and wherein the conjugation of element A to element B results in increased peptide or protein expression when compared to element A lacking element B.
  • the RNA conjugate upon administration to a cell, results in expression levels that are increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to element A lacking element B.
  • the RNA conjugate upon administration to a cell, results in expression levels that are increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to element A lacking element B.
  • a 2-fold e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,
  • element A is an mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the conjugation of element A to element B results in increased half-life of the RNA conjugate when compared to element A lacking element B.
  • half-life (T%) relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
  • the half-life of the RNA conjugate is indicative of the stability of said RNA conjugate.
  • the RNA conjugate has a half-life measurement that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • a half-life measurement that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • the RNA conjugate has a half-life measurement that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 1 00-fold,) when compared to element A lacking element B.
  • a half-life measurement that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 1 00-fold,) when compared to element A lacking element B.
  • the RNA conjugate results in a longer half-life compared to administration of the corresponding element A lacking element B, wherein the additional duration of half-life in said cell, tissue, or organism is at least 5h, 10h, 20h, 25h, 30h, 35h, 40h, 45h, 50h, 55h, 60h, 65h, 70h, 75h, 80h, 85h, 90h, 95h, or 100h or even longer.
  • the additional half-life is about 20h to about 240h.
  • the additional half-life is observed in liver cells.
  • the additional half-life is observed in adipocytes.
  • the additional half-life is observed in muscle cells.
  • element A is an mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the conjugation of element A to element B results in increased resistance to degradation of the RNA conjugate when compared to element A lacking element B.
  • RNA degradation systems e.g. intracellular RNA- degrading enzymes like endonucleases that cut RNA internally, 5’ exonucleases that hydrolyze RNA from the 5’ end, and 3’ exonucleases that degrade RNA from the 3’ end.
  • the RNA conjugate results in resistance to degradation that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to element A lacking element B.
  • the RNA conjugate results in resistance to degradation that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to element A lacking element B.
  • 2-fold to about 100-fold e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,
  • element A is an mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the RNA molecule of element A comprises at least one coding sequence encoding at least one peptide or protein, wherein the conjugation of element A to element B results in increased translation of the peptide or protein when compared to element A lacking element B.
  • the translation of an mRNA is initiated by a set of initiation factor proteins binding to the mRNA including elF4E, elF4G, elF4A, and PABP.
  • This complex promotes the joining of the small subunit of the ribosome which starts scanning the mRNA 5’ to 3’ forthe AUG start codon.
  • the large ribosomal subunit joins, and translation of the open reading frame starts.
  • the RNA conjugate upon administration to a cell, results in translation that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to the element A lacking element B.
  • about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to the element A lacking element B.
  • the RNA conjugate upon administration to a cell, results in translation that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to the element A lacking element B.
  • a 2-fold e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,
  • element A is an mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the conjugation of element A to element B results in reduced induction of innate immune response, e.g. reduced induction of proinflammatory cytokines, when compared to the element A lacking element B.
  • the term “reduced induction of innate immune response” relates to immunostimulatory properties defined as the induction or activation or stimulation of an innate immune response which is determined by measuring the induction of cytokines, preferably proinflammatory cytokines. Reducing the induction of innate immune response is characterized by a reduced level of at least one cytokine preferably selected from MIG, McP1 , Rantes, MIP-1 alpha, IP-10, MIP-1 beta, McP1 , TNFalpha, IFNgamma, IFNalpha, IFNbeta, IL-12, IL-6, or IL-8.
  • the induction of cytokines is measured after administration of the RNA conjugate to cells, a tissue or an organism, preferably hPBMCs, Hela cells or HEK cells. Preferred in that context are hPBMCs.
  • hPBMCs a tissue or an organism
  • an assay for measuring cytokine levels is performed. Cytokines secreted into culture media or supernatants can be quantified by techniques such as bead-based cytokine assays (e.g. cytometric bead array (CBA), ELISA, and Western blot).
  • bead-based cytokine assays e.g. cytometric bead array (CBA), ELISA, and Western blot.
  • the RNA conjugate upon administration to a cell, results in reduced level of at least one cytokine preferably selected from MIG, McP1 , Rantes, MIP-1 alpha, IP-10, MIP-1 beta, McP1 , TNFalpha, IFNgamma, IFNalpha, IFNbeta, IL-12, IL-6, or IL-8, wherein the reduced level of at least one cytokine is a reduction by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • cytokine preferably selected from MIG, McP1 , Rantes, MIP-1 alpha, IP-10, MIP-1 beta, McP1 , TNFalpha
  • the RNA conjugate upon administration to a cell, results in reduced level of at least one cytokine preferably selected from MIG, McP1 , Rantes, MIP-1 alpha, IP-10, MIP-1 beta, McP1 , TNFalpha, IFNgamma, IFNalpha, IFNbeta, IL-12, IL-6, or IL-8, wherein the at least one cytokine is reduced by about 2-fold to about 100-fold (e.g., about 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to element A lacking element B.
  • at least one cytokine preferably selected from MIG, McP1 , Rantes, MIP-1 alpha, IP-10, MIP-1 beta, Mc
  • element A is a mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the RNA molecule of element A comprises at least one cds encoding at least one peptide or protein, wherein the conjugation of element A to element B results in increased presentation of the encoded peptide or protein when compared to element A lacking element B.
  • presentation of the encoded protein or peptide relates to higher amount and/or variety of immunogenic and/or stable peptides presented via MHC class I and II molecules after protein degradation by the proteasomal machinery, and thus cellular immunity, e.g. T cell activation based thereon.
  • the presentation of the encoded protein or peptide on MHC class I and II molecules is increased on cells, comprising immune cells (e.g., T cells), antigen-presenting cells (e.g., dendritic cells, macrophages, engineered antigen-presenting cells), MHC class l-expressing cells, MHC class II- expressing cells, or any combination thereof.
  • the RNA conjugate upon administration to a cell, results in presentation of the encoded protein or peptide that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to element A lacking element B.
  • the RNA conjugate upon administration to a cell, results in presentation of the encoded protein or peptide that is increased by about 2-fold to about 100-fold (e.g., about 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to element A lacking element B.
  • a 2-fold e.g., about 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,
  • element A is mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the invention provides an RNA conjugate, wherein the RNA molecule of element A comprises at least one cds encoding at least one peptide or protein, wherein the conjugation of element A to element B results in increased immunogenicity of the encoded protein or peptide when compared to the element A lacking element B.
  • the effect may be cause by a higher stability and half-life of the RNA conjugate.
  • immunogenicity relates to cellular and humoral immunity, against the encoded protein or peptide.
  • the increased immunogenicity of the RNA conjugate is defined but not limited to enhanced cellular immunity, e.g. CD8/CD4 T-cells responses, against the encoded protein or peptide, increased IFNgamma production by CD8+ T cells upon exposure to the encoded protein or peptide, enhanced humoral immunity, e.g. antibody titers, against the encoded protein or peptide, increased variety of antibody species against the encoded protein or peptide or any combination of these.
  • the RNA conjugate upon administration to a cell, results in increased immunogenicity of the encoded protein or peptide that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to element A lacking element B.
  • about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to element A lacking element B.
  • the RNA conjugate upon administration to a cell, results in immunogenicity of the encoded protein or peptide that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to element A lacking element B.
  • about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold, when compared to element A lacking element B.
  • element A is mRNA.
  • the mRNA encodes a therapeutic protein or peptide.
  • the cell is a mammalian cell, preferably a human cell.
  • the RNA conjugate comprises an element A that comprises:
  • a 5'-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3'-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene
  • a poly(A) sequence preferably comprising about 60 to 100 adenosine nucleotides; wherein the sealing element comprises at least one peptide, preferably at least one peptide derived from a Polyadenylate-binding protein-interacting protein (PAIP), preferably from PAIP1 or PAIP2, according to SEQ ID NO: 1 or SEQ ID NO: 2; wherein element B is conjugated to element A via a linker element L, preferably wherein the linker element L comprises the structure of formula I and wherein formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; and n is 0; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • PAIP Polyadenylate-binding protein-interacting protein
  • the RNA conjugate has an increased resistance to degradation and an increased translation efficiency of a recombinant protein in a cell, preferably a mammalian cell such as a human cell, wherein resistance to degradation and translation efficiency of a recombinant protein is increased when compared to an RNA molecule lacking the sealing element, wherein the sealing element comprises at least one peptide, preferably a peptide derived from a Polyadenylate-binding protein-interacting protein (PAIP), preferably from PAIP1 or PAIP2 according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • PAIP Polyadenylate-binding protein-interacting protein
  • the RNA conjugate comprises an element A that comprises:
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene;
  • a poly(A) sequence preferably comprising about 60 to 100 adenosine nucleotides; wherein the sealing element is selected from at least one nucleic acid, preferably comprising 32 modified adenosines, more preferably 32 phosphorothioate-adenosines or 322 -O-methyl-phosphorothioate-adenosines; wherein element B is conjugated to element A via a linker element L, preferably wherein the linker element L comprises the structure of formula I and wherein formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is C6 alkyl or substituted C5 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to
  • the RNA conjugate has an increased resistance to degradation and an increased expression of a recombinant protein in a cell, wherein resistance to degradation and expression of a recombinant protein is increased compared to an RNA molecule lacking the sealing element, wherein the sealing element comprises at least one nucleic acid, preferably 32 modified adenosines, more preferably 32 phosphorothioate- adenosines or 322 -O-methyl-phosphorothioate-adenosines.
  • the RNA conjugate comprises an element A that comprises:
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene
  • a poly(A) sequence preferably comprising about 60 to 100 adenosine nucleotides; wherein the sealing element is selected from at least one oligonucleotide, preferably 4 modified adenosines, more preferably 4 PNA-adenosines; wherein element B is conjugated to element A via a linker element L, preferably wherein the linker element L comprises the structure of formula I and wherein formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; M is a C2 alkyl or substituted C2 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • the RNA conjugate has an increased resistance to degradation and an increased expression of a recombinant protein in a cell, wherein resistance to degradation and expression of a recombinant protein is increased when compared to an RNA molecule lacking the sealing element, wherein the sealing element comprises at least one polynucleotide, preferably 4 modified adenosines, more preferably 4 PNA-adenosines.
  • the RNA conjugate comprises an element A that comprises:
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene
  • a poly(A) sequence preferably comprising about 60 to 100 adenosine nucleotides; wherein the sealing element is selected from at least one small molecule, preferably a D-Glucosamine; wherein element B is conjugated to element A via a linker element L, preferably wherein the linker element L comprises the structure of formula I and wherein formula I is further specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are each, independently, hydrogen or hydroxyl, preferably hydrogen; n is 0; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • the RNA conjugate has an increased resistance to degradation and an increased expression of a recombinant protein in a cell, wherein resistance to degradation and expression of a recombinant protein is increased when compared to an RNA molecule lacking the sealing element, wherein the sealing element comprises at least one small molecule, preferably D-Glucosamine.
  • the invention provides a pharmaceutical composition comprising at least one RNA conjugate as defined in the first aspect.
  • RNA conjugate has to be read on and have to be understood as suitable embodiments of the pharmaceutical composition of the second aspect and vice versa.
  • features and embodiments described in the context of the present aspect may likewise apply to any other aspect of the invention.
  • composition refers to any type of composition in which the specified ingredients (e.g. the RNA conjugate) may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient.
  • the composition may be a dry composition such as a powder, a granule, ora solid lyophilized form. Alternatively, the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed.
  • the RNA conjugate as comprised in the pharmaceutical composition is provided in an amount of about 10ng to about 500pg, in an amount of about 1 pg to about 500pg, in an amount of about 1 pg to about 100pg, specifically, in an amount of about 1 pg, 2pg, 3pg, 4pg, 5pg, 6pg, 7pg, 8pg, 9pg, 10pg, 11 pg, 12pg, 13pg, 14pg, 15pg, 20pg, 25pg, 30pg, 35pg, 40pg, 45pg, 50pg, 55pg, 60pg, 65pg, 70pg, 75pg, 80pg, 85pg, 90pg, 95pg or 100pg.
  • the pharmaceutical composition comprises a plurality or at least more than one RNA conjugates, preferably wherein each RNA conjugate encodes a different protein.
  • the pharmaceutical composition additionally comprises at least one different RNA species, for example an mRNA, a replicon RNA, or a circular RNA.
  • these further RNA species lack element B.
  • the at least one RNA conjugate of the pharmaceutical composition is formulated with a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” as used herein preferably includes the liquid or non-liquid basis of the composition for administration.
  • compositions of the present invention are suitably sterile and/or pyrogen-free.
  • the at least one RNA conjugate of the pharmaceutical composition is complexed or associated with at least one further compound to obtain a formulated composition.
  • a formulation in that context may have the function of a transfection agent.
  • a formulation in that context may also have the function of protecting the RNA conjugate from degradation, e.g. to allow storage, shipment, etc.
  • the at least one RNA conjugate of the pharmaceutical composition is formulated with at least one compound, e.g. peptides, proteins, lipids, polysaccharides, and/or polymers.
  • the at least one RNA conjugate of the pharmaceutical composition is formulated with at least one cationic (cationic or preferably ionizable) or polycationic compound (cationic or preferably ionizable).
  • the at least one RNA conjugate of the pharmaceutical composition is complexed or associated with or at least partially complexed or partially associated with one or more cationic (cationic or preferably ionizable) or polycationic compound.
  • cationic or polycationic compound as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a charged molecule, which is positively charged at a pH value ranging from about 1 to 9, at a pH value ranging from about 3 to 8, at a pH value ranging from about 4 to 8, at a pH value ranging from about 5 to 8, more preferably at a pH value ranging from about 6 to 8, even more preferably at a pH value ranging from about 7 to 8, most preferably at a physiological pH, e.g. ranging from about 7.2 to about 7.5.
  • a cationic component e.g.
  • a cationic peptide, cationic protein, cationic polymer, cationic polysaccharide, cationic lipid may be any positively charged compound or polymer which is positively charged under physiological conditions. Accordingly, “polycationic” components are also within the scope exhibiting more than one positive charge under the given conditions.
  • the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
  • the at least one RNA conjugate is formulated in lipid-based carriers.
  • lipid-based carrie encompasses lipid-based delivery systems for RNA that comprise a lipid component.
  • a lipid-based carrier may additionally comprise other components suitable for formulating the RNA conjugate including a cationic or polycationic polymer, polysaccharide, protein, peptide, or any combinations thereof.
  • RNA conjugate completely or partially be incorporated or encapsulated in a lipid-based carrier, wherein the RNA conjugate may be located in the interior space of the lipid-based carrier, within the lipid layer/membrane of the lipid- based carrier, or associated with the exterior surface of the lipid-based carrier.
  • encapsulation The incorporation of nucleic acid into lipid- based carriers may be referred to as "encapsulation”.
  • encapsulation refers to the essentially stable combination an RNA with one or more lipids into larger complexes or assemblies such as lipid-based carriers, preferably without covalent binding of the nucleic acid.
  • the encapsulated RNA may be completely or partially located in the interior of the lipid-based carrier (e.g. the lipid portion and/or an interior space) and/or within the lipid layer/membrane of the lipid-based carriers.
  • the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polylpexes, and/or nanoliposomes.
  • the lipid-based carriers are lipid nanoparticles (LNPs).
  • the lipid nanoparticles encapsulate the at least one RNA conjugate of the invention.
  • LNPs are microscopic lipid particles having a solid or partially solid core. Typically, an LNP does not comprise an interior aqua space sequestered from an outer medium by a bilayer.
  • the RNA may be encapsulated in the lipid portion of the LNP, enveloped by some or the entire lipid portion of the LNP.
  • An LNP may comprise any lipid capable of forming a particle to which the nucleic acid such as the RNA may be attached, or in which RNA may be encapsulated.
  • the lipid-based carriers preferably the LNPs, comprise at least one or more lipids selected from at least one aggregation-reducing lipid, at least one cationic lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog, or any combinations thereof.
  • the lipid-based carriers of the pharmaceutical composition comprise (i) at least one aggregation-reducing lipid, (ii) at least one cationic lipid or ionizable lipid, (iii) at least one neutral lipid or phospholipid, (iv) and at least one steroid or steroid analog.
  • the lipid-based carriers comprise at least one cationic or ionizable lipid.
  • the cationic or ionizable lipid may be cationisable or ionizable, i.e. it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the cationic or ionizable lipid preferably carries a net positive charge at physiological pH, more preferably the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group. Accordingly, in preferred embodiments, the lipid-based carriers comprise a cationic or ionizable lipid selected from an amino lipid.
  • the at least one cationic or ionizable lipid is selected from lipids disclosed in WO2018078053 (i.e. lipids derived from formula I, II, and III, or lipids as specified in claims 1 to 12), the disclosure of WO2018078053 hereby incorporated by reference.
  • lipids disclosed in Table 7 of WO2018078053 e.g. lipids derived from formula 1-1 to 1-41
  • lipids disclosed in Table 8 of WO2018078053 e.g. lipids derived from formula 11-1 to II-36
  • formula 1-1 to formula 1-41 and formula 11-1 to formula II-36 of WO2018078053A, and the specific disclosure relating thereto, are herewith incorporated by reference.
  • the at least one cationic or ionizable lipid is selected or derived from structures 111-1 to III-36 of Table 9 of WO2018078053. Accordingly, formula 111-1 to HI-36 of WO2018078053, and the specific disclosure relating thereto, are herewith incorporated by reference.
  • the at least one cationic or ionizable lipid is selected or derived from formula HI-3 of WD2018078053.
  • a preferred lipid of said formula HI-3 has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate), also referred to as ALC-0315, i.e. CAS Number 2036272-55-4.
  • suitable cationic lipids may be selected or derived from cationic lipids according to PCT claims 1 to 14 of WD2021123332, or table 1 of WO2021123332, the disclosure relating thereto herewith incorporated by reference. Accordingly, suitable cationic lipids may be selected or derived from cationic lipids according to Compound 1 to Compound 27 (C1-C27) of Table 1 of WO2021123332.
  • the at least one cationic or ionizable lipid is selected or derived from SS-33/4PE-15 (see C23 in Table 1 of WD2021123332). In other embodiments, the at least one cationic or ionizable lipid is selected or derived from HEXA- C5DE-PipSS (see C2 in Table 1 of WO2021123332). In other embodiments, the at least one cationic or ionizable lipid is selected or derived from compound C26 (VitE-C4DE-Pip-thioether) as disclosed in Table 1 of WD2021123332.
  • the at least one cationic or ionizable lipid is selected or derived from 9-Heptadecanyl 8- ⁇ (2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate, also referred to as SM-102, i.e. CAS Number 2089251 -47-6.
  • the at least one cationic or ionizable lipid is selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS, or compound C26 (see C26 in Table 1 of WO2021123332).
  • the lipid-based carriers comprise at least one neutral lipid or phospholipid.
  • neutral lipid refers to any lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.
  • Neutral lipids may be selected from DHPC, DHPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-Omonomethyl PE, 16-Odimethyl PE, 18-1 -trans PE, SOPE, transDOPE, 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), DPhyPS (1 ,2-diphytanoyl-sn-glycero-3- phospho-L-serine), or mixtures thereof.
  • DPhyPE DPhyPS (1 ,2-diphytanoyl-sn-glycero-3- phospho-L-serine
  • the at least one neutral lipid is selected or derived from DSPC, DHPC, DPhyPE, or DPhyPS.
  • Steroids, steroid analogues or sterols Steroids, steroid analogues or sterols:
  • the lipid-based carriers comprise a steroid, steroid analog or sterol.
  • the steroid or steroid analog is selected or derived from cholesterol, cholesteryl hemisuccinate (CHEMS), preferably cholesterol.
  • the lipid-based carriers comprise at least one aggregation reducing lipid or moiety.
  • aggregation reducing moiety refers to a molecule comprising a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
  • aggregation reducing lipid refers to a molecule comprising both a lipid portion and a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
  • the lipid-based carriers may undergo charge-induced aggregation, a condition which can be undesirable forthe stability of the lipid-based carriers. Therefore, it can be desirable to include a compound or moiety which can reduce aggregation, for example by sterically stabilizing the lipid-based carriers.
  • Such a steric stabilization may occur when a compound having a sterically bulky but uncharged moiety that shields or screens the charged portions of a lipid-based carriers from close approach to other lipid-based carriers in the composition.
  • stabilization of the lipid-based carriers is achieved by including lipids which may comprise a lipid bearing a sterically bulky group which, after formation of the lipid-based carrier, is preferably located on the exterior of the lipid- based carrier.
  • Suitable aggregation reducing groups include hydrophilic groups, e.g. monosialoganglioside GM1 , polyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g., polyethylene glycol) or polypropylene glycol).
  • the aggregation reducing lipid is a polymer conjugated lipid.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion, wherein the polymer is suitable of reducing or preventing aggregation of lipid-based carriers comprising the RNA conjugate.
  • a polymer has to be understood as a substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits.
  • a suitable polymer in the context of the invention may be a hydrophilic polymer.
  • An example of a polymer conjugated lipid is a PEGylated or PEG-conjugated lipid.
  • the polymer conjugated lipid is selected from a PEG-conjugated lipid or a PEG-free lipid.
  • the polymer conjugated lipid is a PEG-conjugated lipid (or PEGylated lipid, PEG lipid).
  • the average molecular weight of the PEG moiety in the PEG-conjugated lipid preferably ranges from 500 to 8,000 Daltons (e.g., from 1 ,000 to 4,000 Daltons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Daltons.
  • the polymer conjugated lipid e.g.
  • the PEG-conjugated lipid is selected or derived from 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG 2000), C10- PEG2K, or Cer8-PEG2K.
  • the polymer conjugated lipid is selected or derived from formula (IV) of WD2018078053, preferably selected from formula (IVa) of WD2018078053.
  • a preferred polymer conjugated lipid is selected from 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.
  • the aggregation reducing lipid is selected from a PEG-free lipid, e.g. a PEG-free polymer conjugated lipid.
  • the aggregation reducing lipid is a PEG-free lipid that comprises a polymer different from PEG.
  • a PEG-free polymer conjugated lipid may be selected or derived from a “POZ-lipid”.
  • POZ lipid or respectively preferred polymer conjugated lipids are described in WD2023031394, the full disclosure herewith incorporated by reference.
  • disclosure relating to polymer conjugated lipids as defined in any one of claims 1 to 8 of WO2023031394 are incorporated by reference.
  • the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid.
  • the polymer conjugated lipid is selected or derived from PMOZ 1 , PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 of W02023031394.
  • the polymer conjugated lipid is selected or derived from PMOZ 4 according to formula “PMOZ 4” ofWO2023031394.
  • the at least one aggregation-reducing lipid is selected or derived from DMG- PEG 2000, C10-PEG2K, Cer8-PEG2K, a POZ-lipid such as PMOZ4, or ALC-0159.
  • Lipid-based carrier compositions are Lipid-based carrier compositions:
  • the lipid-based carriers comprise at least one RNA conjugate as defined herein, a cationic or ionizable lipid as defined herein, an aggregation reducing lipid as defined herein, a neutral lipid as defined herein, and, a steroid or steroid analog as defined herein.
  • the lipid-based carriers comprising the at least one RNA conjugate comprise
  • the lipid-based carrier preferably the LNP, comprising the RNA conjugate, comprise
  • At least one cationic lipid selected or derived from ALC-0315, SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS or C26;
  • the lipid-based carrier preferably the LNP, comprises (i) to (iv) in a molar ratio of about 20-60% cationic lipid or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid e.g. polymer conjugated lipid.
  • the lipid-based carrier preferably the LNP, comprise (i) to (iv) in a molar ratio of about 45- 60% cationic lipid or ionizable lipid, about 5-15% neutral lipid, about 25-45% steroid or steroid analog, and about 0.5- 2.5% aggregation reducing lipid e.g. polymer conjugated lipid.
  • the lipid-based carrier preferably the LNP, comprising RNA conjugate, comprise
  • a cationic lipid ALC-0315 (i) a neutral lipid DSPC; (iii) a steroid or steroid analog cholesterol; and (iv) an aggregation reducing lipid ALC-0159; preferably wherein (i) to (iv) are in a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1 .7% aggregation reducing lipid, preferably wherein the lipid- based carrier encapsulates the RNA conjugate.
  • the amount of lipid comprised in the lipid-based carriers may be selected taking the amount of the RNA conjugate cargo into account. In one embodiment, these amounts are selected such as to result in an N/P ratio of the lipid-based carriers encapsulating the RNA conjugate in the range of about 0.1 to about 20.
  • the N/P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid to the phosphate groups (“P”) of the RNA conjugate, which is used as cargo.
  • the N/P ratio may be calculated on the basis that, for example, 1 pg RNA conjugate typically contains about 3nmol phosphate residues, provided that the RNA conjugate exhibits a statistical distribution of bases.
  • the “N”-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and - if present - cationisable groups.
  • the N/P ratio can be in the range of about 1 to about 50. In other embodiments, the range is about 5 to about 20, e.g. about 6 or about 14 or about 17.
  • the pharmaceutical composition comprises lipid-based carriers (encapsulating RNA conjugate) that have a defined size (particle size, homogeneous size distribution).
  • the size of the lipid-based carriers such as LNPs is typically described herein as Z-average size.
  • Z-average size refers to the mean diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless.
  • DLS dynamic light scattering
  • PI polydispersity index
  • the lipid-based carriers preferably the LNPs, have a Z-average size ranging from about 50nm to about 200nm, preferably from about 50nm to about 150nm, more preferably from about 50nm to about 120nm.
  • the pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% lipid-based carriers that have a particle size exceeding about 500nm.
  • the pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% LNPs that have a particle size smallerthan about 20nm.
  • at least about 80%, 85%, 90%, 95% of lipid-based carriers have a spherical morphology.
  • the polydispersity index (PDI) of the lipid-based carriers is typically in the range of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2. Typically, the PDI is determined by dynamic light scattering.
  • RNA conjugates are encapsulated in a lipid-based carrier such as an LNP.
  • the percentage of encapsulation may be determined by a RiboGreen assay as known in the art.
  • the invention provides a combination of RNA conjugates and at least one different RNA species.
  • embodiments relating to the RNA conjugate of the first aspect may likewise be read on and be understood as suitable embodiments of the combination comprising RNA conjugate of the third aspect.
  • embodiments relating to the pharmaceutical composition of the second aspect may likewise be read on and be understood as suitable embodiments of the combination of the third aspect and vice versa.
  • the term “combination” preferably means a combined occurrence of the at least one RNA conjugate (herein referred to as “component I.”), and of the at least one different RNA species that lacks element B (herein referred to as “component II.’). Therefore, said combination may occur either as one composition (as outlined e.g. in the context of the second aspect), comprising all these components in one and the same composition or mixture (but as separate entities), or may occur as a kit of parts, wherein the different components form different parts of such a kit of parts (as outlined e.g. in the context of the fourth aspect).
  • the administration of the components of the combination may occur either simultaneously or timely staggered, either at the same site of administration or at different sites of administration, as further outlined below.
  • the components may be formulated together as a co-formulation or may be formulated as different separate formulations (and optionally combined after formulation).
  • RNA conjugate of the invention may be combined with a different RNA species, preferably a coding RNA, as e.g. both molecules may have different translation profiles.
  • component II comprises at least one coding RNA preferably selected from an mRNA, a replicon RNA, or a circular RNA.
  • these coding RNA species lack element B as specified herein.
  • the combination comprises an RNA conjugate encoding a therapeutic peptide or protein and a coding RNA encoding essentially the same therapeutic peptide or protein.
  • RNA conjugate may lead to a fast protein translation (e.g. provided by the coding RNA) and a long-lasting protein translation (provided by the RNA conjugate).
  • the combination upon administration of the combination to a cell or subject, has reduced immunostimulatory properties (as defined herein) compared to an administration of component II alone.
  • the combination has at least 10%, 20% or at least 30% lower immunostimulatory properties (as defined herein) compared to component II alone.
  • the combination has at least 40%, 50% or at least 60% lower immunostimulatory properties (as defined herein) compared to component II alone.
  • the combination upon administration of the combination to a cell or subject, has a prolonged protein expression (that is an additional duration of protein expression) compared to an administration of component II alone.
  • the additional duration of protein expression is at least 5h, 10h, 20h, 25h, 30h, 35h, 40h, 45h, 50h, 55h, 60h, 65h, 70h, 75h, 80h, 85h, 90h, 95h, or 100h or even longer.
  • the additional duration of protein expression is about 20h to about 240h.
  • the combination upon administration of the combination to a cell or subject, has a faster onset of protein expression compared to an administration of component I alone.
  • the faster onset of protein expression is to be understood as a detectable protein expression that starts at least 1 h, 2h, 3h, 4h, 5h, 5h, 10h, 20h, 25h earlier.
  • the faster onset of protein expression is to be understood as the peak protein expression that is achieved at least 1 h, 2h, 3h, 4h, 5h, 5h, 10h, 20h, 25h earlier.
  • the administration of the components of the combination may occur either simultaneously or timely staggered, either at the same site of administration or at different sites of administration.
  • administration of the combination is intravenous, intranasal, intramuscular, intradermal, transdermal, intraocular, subcutaneous, intrapulmonal, intralesional, intrathecal, intracranial, intracardial or intratumoral.
  • the invention provides a kit or kit of parts comprising at least one RNA conjugate of the first aspect, and/or at least one pharmaceutical composition of the second aspect, and/or at least one combination of the third aspect.
  • RNA conjugate of the first aspect
  • pharmaceutical composition of the second aspect
  • combination of the third aspect.
  • embodiments relating to the RNA conjugate, the pharmaceutical composition, orthe combination may likewise be read on and be understood as suitable embodiments of the kit or kit of parts of the fourth aspect and vice versa.
  • the kit or kit of parts may comprise a liquid vehicle for solubilizing, and/or technical instructions providing information on administration and dosage of the components.
  • the technical instructions of said kit may contain information about administration and dosage and patient groups.
  • kits, preferably kits of parts may be applied e.g. for any of the applications or uses mentioned herein, preferably for the use of the RNA conjugate, the pharmaceutical composition, orthe combination for the treatment or prophylaxis of diseases, disorder, or condition.
  • the kit or kit of parts as defined herein comprises at least one syringe or application device.
  • the present invention relates to the medical use of the at least one RNA conjugate as defined herein, the pharmaceutical composition as defined herein, the combination as defined herein, or the kit or kit of parts as defined herein.
  • embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of medical uses of the invention.
  • embodiments relating to medical uses as described herein of course also relate to methods of treatments.
  • the invention provides an RNA conjugate of the first aspect, or a pharmaceutical composition of the second aspect, or a combination of the third aspect, or a kit or kit of the fourth aspect, for use as a medicament in treating or preventing a disease, disorder, or condition in a subject.
  • the invention provides an RNA conjugate, a pharmaceutical composition (comprising the RNA conjugate), a combination (comprising the RNA conjugate), or the kit or kit of parts (comprising the RNA conjugate), wherein element A of the RNA conjugate comprises at least one RNA molecule as defined herein, and element B comprises a sealing element selected from at least one peptide, preferably a peptide derived from a PAIR protein, preferably according to SEQ ID NO: 1 or 2 for use as a medicament in treating or preventing a disease, disorder, or condition in a subject.
  • the RNA conjugate encodes a peptide that comprises an amino acid sequence being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 2, or a fragment or variant thereof.
  • the invention provides an RNA conjugate, a pharmaceutical composition (comprising the RNA conjugate), a combination (comprising the RNA conjugate), or the kit or kit of parts (comprising the RNA conjugate), wherein element A of the RNA conjugate comprises at least one RNA molecule as defined herein, and element B comprises a sealing element selected from at least one polynucleotide, preferably 32 modified adenosines, more preferably 322‘-0-methyl-adenosines phosphorothioate or 322‘-0-methyl-adenosines for use in treating or preventing a disease, disorder, or condition in a subject.
  • the RNA conjugate comprises a polynucleotide sequence being identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 14, 15, 16 or 17, ora fragment or variant thereof.
  • the invention provides an RNA conjugate, a pharmaceutical composition (comprising the RNA conjugate), a combination (comprising the RNA conjugate), or the kit or kit of parts (comprising the RNA conjugate), wherein element A of the RNA conjugate comprises at least one RNA molecule as defined herein, and element B comprises a sealing element selected from at least one oligonucleotide, preferably 4 modified adenosines, more preferably 4 PNA-adenosines for use in treating or preventing a disease, disorder, or condition in a subject.
  • the RNA conjugate comprises an oligonucleotide sequence being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to AAAA, wherein the adenosines are PNA-adenosines, or a fragment or variant thereof.
  • the invention provides an RNA conjugate, a pharmaceutical composition (comprising the RNA conjugate), a combination (comprising the RNA conjugate), or the kit or kit of parts (comprising the RNA conjugate), wherein element A of the RNA conjugate comprises at least one RNA molecule as defined herein, and element B comprises a sealing element selected from at least one small molecule, preferably D-Glucosamine for use in treating or preventing a disease, disorder, or condition in a subject.
  • the RNA conjugate comprises a molecular structure being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to formula IV, or a fragment or variant thereof.
  • the use may be for human or veterinary purposes, preferably for human medical purposes.
  • the use may be for human medical purposes, in particular for young infants, new-borns, immunocompromised recipients, pregnant and breast-feeding women, and elderly people.
  • the RNA conjugate, the pharmaceutical composition, the combination, orthe kit or kit of parts is administered by intramuscular, intranasal, transdermal, intradermal, intralesional, intracranial, subcutaneous, intracardial, intratumoral, intravenous, intrapulmonal, intraarticular, sublingual, pulmonary, intrathecal, or ocular administration.
  • the present invention provides an RNA conjugate of the first aspect, or a pharmaceutical composition of the second aspect, or a combination of compound of the third aspect, or a kit or kit of parts of the fourth aspect, for use in treating or preventing an infectious disease, a tumour disease, or a genetic disease, disorder or condition.
  • the present invention provides an RNA conjugate of the first aspect, or a pharmaceutical composition of the second aspect, or a combination of compound of the third aspect, or a kit or kit of parts of the fourth aspect, for use in treating or preventing an infectious disease, a tumour disease, or a genetic disease, disorder or condition, where a long-term expression of the target peptide or protein is necessary.
  • the present invention relates to a method of treating or preventing a disease, disorder or condition.
  • embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of method of treatments of the invention.
  • specific features and embodiments relating to method of treatments as provided herein may also apply for medical uses of the invention and vice versa.
  • Preventing (Inhibiting) or treating a disease, in particular a virus infection relates to inhibiting the frill development of a disease or condition, for example, in a subject who is at risk for a disease such as a virus infection.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating”, with reference to a disease or pathological condition refers to any observable beneficial effect of the treatment.
  • Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or wellbeing of the subject, or by other parameters that are specific to the particular disease.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • the present invention relates to a method of treating or preventing a disease, disorder or condition, wherein the method comprises applying or administering to a subject in need thereof the RNA conjugate of the first aspect, the pharmaceutical composition of the second aspect, the combination of the third aspect, or the kit or kit of parts of the fourth aspect.
  • the disease, disorder or condition is an infectious disease, a tumour disease, or a genetic disease, disorder or condition.
  • the subject in need is a mammalian subject, preferably a human subject, e.g. new-bom human subject, pregnant human subject, immunocompromised human subject, and/or elderly human subject.
  • a mammalian subject preferably a human subject, e.g. new-bom human subject, pregnant human subject, immunocompromised human subject, and/or elderly human subject.
  • the applying or administering is performed via intramuscular injection, intradermal injection, transdermal injection, intradermal injection, intralesional injection, intracranial injection, subcutaneous injection, intracardial injection, intratumoral injection, intravenous injection, intraocular injection, intrapulmonal inhalation, intraarticular injection, or sublingual injection.
  • the present invention relates to a method producing an RNA conjugate.
  • the method of producing an RNA conjugate comprises the steps of a. Providing an RNA molecule (as element A) encoding at least one protein or peptide as defined herein, preferably encoding a therapeutic peptide or protein as defined herein; b. Purifying the RNA molecule by at least one step of purification; c. Oxidizing the purified RNA molecule of step b) with an oxidant, preferably sodium periodate, resulting in oxidative ring opening of the sugar at the 3’-terminus of the RNA into a dialdehyde; d.
  • an oxidant preferably sodium periodate
  • RNA conjugate Condensation of the oxidized RNA molecule with a nucleophile, preferably wherein the nucleophile is provided by an element B, more preferably a sealing element as defined in the context of the first aspect; e.
  • element A is conjugated to element B by stepwise solid-phase conjugation, synthetic conjugation or post-synthetic conjugation.
  • the post-synthetic conjugation comprises a solid phase conjugation or in solution conjugation.
  • the post synthetic conjugation is an in-solution conjugation.
  • element A, the RNA molecule has been conjugated to element B via reductive amination of periodate-oxidized RNA.
  • the sealing element used in step d) comprises a nucleophile group preferably provided by the linker element L as defined herein.
  • element C (the sealing element) comprises a nucleophile group as defined herein.
  • the nucleophile group C is an amine group or activated amine group.
  • element C (the sealing element) comprises element NH2-(M)n- to provide the amine group or activated amine group for conjugation.
  • element C (the sealing element) comprises the element NH2-(M)n- to enable conjugation to element A preferably via element (see e.g. reaction scheme 2 of Example 1 .3).
  • element C (the sealing element) comprises element NH2-(M)n-, wherein (M)n comprises O, NR N , a bond, optionally substituted C1 -C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted diethylen glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted C2-C100 polyethylene glycol, or optionally substituted C1 -C30 heteroalkylene, heteroalkenylene or heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl; and wherein n is 0, 1 , 2, 3, 4, or 5.
  • element C (the sealing element) comprises NH2-(M)n-, wherein (M)n comprises a bond, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, substituted C5 alkyl or C6 alkyl, and wherein n is 1 .
  • the providing step a) comprises a step of RNA in vitro transcription of a DNA template encoding the RNA molecule.
  • the providing step a) comprises a step of co-transcriptional capping using a cap analog, preferably a modified cap1 analogue, more preferably a cap analogue that lacks a diol group.
  • the method additionally comprises a step of enzymatic capping that is preferably carried out after step e).
  • the at least one step of purification carried out in step b) and/or f) is selected from RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flowthrough chromatography, oligo(dT) purification, affinity chromatography, cellulose-based purification, or any combination thereof.
  • the at least one step of purification carried out in step f) is an affinity chromatography selective for element B.
  • the method is for producing an RNA conjugate that is characterized by any one of the features as presented in the context of the first aspect.
  • the method additionally comprises a step of formulating the obtained RNA conjugate preferably into lipid-based carriers, suitably in lipid-based carriers such as LNPs that are characterized by any one of the features as presented in the context of the second aspect.
  • lipid-based carriers such as LNPs
  • the present invention relates to the use of a element C (the sealing element).
  • an element C is for increasing or prolonging half-life of an RNA molecule, increasing resistance to degradation of an RNA molecule, increasing or prolonged stability of an RNA molecule, increasing expression of an RNA molecule, reducing induction of innate immune response of an RNA molecule, reducing induction of proinflammatory cytokines and/or an increasing translation efficiency of an RNA molecule.
  • element C is selected from at least one peptide as defined herein, protein as defined herein, nucleoside as defined herein, nucleotide as defined herein, nucleic acid as defined herein, targeting moiety as defined herein, small molecule as defined herein, or polymer as defined herein.
  • the present invention relates to a method of increasing or prolonging protein expression of an RNA molecule
  • the method of increasing or prolonging protein expression of an RNA molecule comprises a step of conjugating a sealing element (element B) as defined herein to an RNA molecule (element A) to generate an RNA conjugate of the invention and applying or administering the RNA conjugate to a cell or a subject.
  • generating of the RNA conjugate is carried out using methods as specified herein.
  • the invention provides a method of increasing the expression of a recombinant peptide or protein of interest in a cell comprising contacting the cell with the RNA conjugate, and wherein expression is increased when compared to element A lacking element B.
  • the invention provides a method of increasing the half-life of the RNA conjugate of interest in a cell comprising contacting the cell with the RNA conjugate, and wherein half-life is increased when compared to element A lacking element B.
  • the invention provides a method of increasing the resistance to degradation of an RNA conjugate in a cell comprising contacting the cell with the RNA conjugate comprising element A, a linker and at least one element B, and wherein resistance to degradation is increased when compared to element A lacking element B.
  • the invention provides a method of increasing the translation of a recombinant peptide or protein of interest in a cell comprising contacting the cell with the RNA conjugate and wherein translation is increased when compared to element A lacking element B.
  • the invention provides a method of reducing the induction of innate immune response, e.g. reduced induction of proinflammatory cytokines, of an RNA conjugate in a cell comprising contacting the cell with the RNA conjugate and wherein induction of immune response, e.g. reduced induction of proinflammatory cytokines, is reduced when compared to element A lacking element B.
  • induction of immune response e.g. reduced induction of proinflammatory cytokines
  • the invention provides a method of increasing the presentation of a recombinant peptide or protein of interest in a cell comprising contacting the cell with the RNA conjugate and wherein presentation of the encoded protein or peptide is increased when compared to element A lacking element B.
  • the invention provides a method of increased immunogenicity of the encoded protein or peptide of interest in a cell comprising contacting the cell with the RNA conjugate and wherein immunogenicity of the encoded protein or peptide is increased when compared to element A lacking element B.
  • Item 1 An RNA conjugate comprising at least one element A and at least one element B, wherein element A comprises or consists of an RNA molecule comprising at least one coding sequence encoding at least one peptide or protein, and wherein element B comprises or consists of at least one sealing element.
  • Item 2A The RNA conjugate of item 1 , wherein the at least one sealing element is element C.
  • Item 2B The RNA conjugate of item 1 , wherein element B is conjugated to element A via a linker element L.
  • Item 3 The RNA conjugate of item 1 or 2, wherein the sealing element is selected from at least one peptide, protein, nucleoside, nucleotide, nucleic acid, targeting moiety, small molecule, or polymer.
  • Item 4 The RNA conjugate of items 1 to 3, wherein the sealing element has at least one of the following functions selected from increasing or prolonging half-life, increasing resistance to degradation, increasing or prolonged stability, increasing expression, reducing induction of innate immune response, reducing induction of proinflammatory cytokines and/or an increasing translation efficiency when introduced into a population of cells.
  • Item 5 The RNA conjugate of items 1 to 4, wherein element B comprises at least two or more sealing elements.
  • Item 6 The RNA conjugate of items 1 to 5, wherein the sealing element comprises or consists of at least one peptide.
  • Item 7 The RNA conjugate of item 6, wherein the at least one peptide comprises about 1 to about 50 amino acids, about 10 to about 50 amino acids, preferably about 15 to 20 amino acids.
  • Item 8 The RNA conjugate of item 6 or 7, wherein the at least one peptide is selected from or derived from a coactivator in the regulation of translation initiation, a peptide regulating mRNA degradation and stability, a nuclear localization peptide, an ER localization peptide, an enhancer of an antigen-presentation peptide, an endosomal escape peptide, an immune stimulation peptide, a Golgi apparatus localization peptide, a lysosomal localization peptide, a mitochondrial localization peptide, and/or a peptide for purification or affinity chromatography, or a fragment or variant of any of these, preferably from a coactivator in the regulation of translation initiation, or a fragment or variant thereof.
  • Item 9 The RNA conjugate of items 6 to 8, wherein the at least one peptide is selected from a coactivator in the regulation of translation initiation selected from or derived from a Polyadenylate-binding protein-interacting protein (PAIP), preferably from PAIP1 or PAIP2, or a fragment or variant thereof.
  • PAIP Polyadenylate-binding protein-interacting protein
  • Item 10 The RNA conjugate of items 6 to 9, wherein the at least one peptide comprises a PABPC1 -interacting motif of a Polyadenylate-binding protein-interacting protein (PAIP), preferably a PABPC1 -interacting motif 1 or 2.
  • PAIP Polyadenylate-binding protein-interacting protein
  • Item 11 The RNA conjugate of items 6 to 10, wherein the at least one peptide comprises at least one amino acid sequence selected or derived from a PAIP1 or PAIP2 protein being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-8, or fragments or variants of any of these.
  • Item 11 b The RNA conjugate of items 6 to 10, wherein the at least one peptide comprises at least one amino acid sequence selected or derived from elF4G being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 145, or fragments or variants thereof.
  • Item 12 The RNA conjugate of items 6 to 11 , wherein the at least one peptide comprises at least one amino acid sequence selected or derived from a PABPC1 -interacting motif of a Polyadenylate-binding proteininteracting protein (PAIP) being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1 or 2, or fragments or variants of any of these.
  • PAIP Polyadenylate-binding proteininteracting protein
  • Item 13 The RNA conjugate of items 1 to 5, wherein the sealing element comprises or consists of at least one nucleoside and/or nucleotide.
  • Item 14 The RNA conjugate of item 13, wherein the at least one nucleoside and/or nucleotide is a modified nucleoside and/or modified nucleotide, preferably L-adenosine, 2’-0-methyl-adenosine, alpha-thio-2 -O- methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine, PNA- adenosine, or inverted thymidine.
  • L-adenosine 2’-0-methyl-adenosine, alpha-thio-2 -O- methyl-adenosine
  • 2’-fluoro-adenosine arabino-adenosine, hexitol-adenosine, LNA-adenosine, PNA- adenosine, or inverted thym
  • Item 15 The RNA conjugate of items 1 to 5, wherein the sealing element comprises or consists of at least one nucleic acid, preferably at least one oligonucleotide or at least one polynucleotide.
  • Item 16 The RNA conjugate of item 15, wherein the at least one nucleic acid comprises about 2 to about 100 nucleotides.
  • Item 17 The RNA conjugate of item 15 or 16, wherein the at least one nucleic acid comprises at least one modified nucleotide or at least two or more modified nucleotides or modification.
  • Item 18 The RNA conjugate of item 17, wherein the modified nucleotide or modification is selected from phosphorothioate, phosphorodithioate, methyl phosphonate, mesyl phosphoramidate, boranophosphate, LNA (locked nucleic acid), 2’-0-methylation (of ribose), 2’-methoxyethoxy (of ribose), methoxyethyl (of ribose), phosphordiamidate morpholino oligomer, 2 -O-methyl-phosphorothioate, 2’-deoxy-2’-a-fluoro, deoxynucleotide, 2’-0-methoxymethyl (of ribose), phosphorodiamidate morpholino oligonucleotides, 2’-O- propargyl, L-ribose, dihydro-base, inverted base, hexitol-base, arabino-sugar, PNA (
  • Item 19 The RNA conjugate items 15 to 18, wherein the at least one nucleic acid comprises at least one modified adenosine, preferably at least one PNA-adenosine, 2 -O-methyl-phosphorothioate adenosine, 2’-O- methyl-adenosine or phosphorothioate-adenosine or a combination thereof.
  • Item 20 The RNA conjugate of items 15 to 19, wherein the at least one nucleic acid comprises a plurality of adenosines, preferably modified adenosines, wherein the plurality of adenosines comprises about 4 to about 40 adenosines.
  • Item 21 The RNA conjugate of item 20, wherein the plurality of adenosines comprises 4 PNA-adenosines, 6 PNA- adenosines, 10 PNA adenosines, 162 -O-methyl-phosphorothioate-adenosines, 32 phosphorothioate- adenosines or 322 -O-methyl-phosphorothioate-adenosines. (SEQ ID NOs: 14-17)
  • Item 22 The RNA conjugate of items 15 to 21 , wherein the at least one nucleic acid comprises at least one antagonist of at least one RNA sensing pattern recognition receptor, preferably a TLR7 antagonist and/or a TLR8 antagonist.
  • Item 23 The RNA conjugate of item 22, wherein the at least one RNA sensing pattern recognition receptor antagonist comprises at least one modified nucleotide, preferably at least one modified guanosine, adenosine or uridine, more preferably at least one 2’-O-methyl guanosine, phosphorothioate-guanosine, 2 -O-methyl-phosphorothioate-guanosine, 2 -O-methyl-phosphorothioate-adenosine, 2’-O-methyl- adenosine, phosphorothioate adenosine, 2’-0-methyl-phosphorothioate-uridine, phosphorothioate uridine or2’-0-methyl-uridine or a combination thereof.
  • the at least one RNA sensing pattern recognition receptor antagonist comprises at least one modified nucleotide, preferably at least one modified guanosine, adenosine or uridine, more preferably at least one 2’
  • Item 24 The RNA conjugate of items 15 to 23, wherein the at least one nucleic acid, preferably the RNA sensing pattern recognition receptor antagonist, comprises or consists of at least one nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NOs: 85-212 of WO2021028439, or fragments or variants of any of these.
  • the at least one nucleic acid preferably the RNA sensing pattern recognition receptor antagonist, comprises or consists of at least one nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NOs: 85-212 of WO2021028439, or fragments or variants of any of these.
  • Item 25 The RNA conjugate of items 15 to 24, wherein the at least one nucleic acid, preferably the at least one RNA sensing pattern recognition receptor antagonist comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence GAGCG(2’OMe)GCCA, SEQ ID NO: 149 or SEQ ID NO: 18.
  • Item 26 The RNA conjugate of items 1 to 5, wherein the sealing element comprises or consists of at least one small molecule.
  • Item 27 The RNA conjugate of item 26, wherein the at least one small molecule comprises a benzyl group, an alkyl group, a sugar, an amino sugar, a heterocycle and/or a heteroatom.
  • Item 28 The RNA conjugate of items 25 or 26, wherein the at least one small molecule is selected from an amino sugar, preferably D-Glucosamine.
  • Item 29 The RNA conjugate of items 1 to 5, wherein the sealing element comprises at least one protein.
  • Item 30 The RNA conjugate of item 29, wherein the at least one protein comprises at least one modified amino acid.
  • Item 31 The RNA conjugate of items 29 or 30, wherein the at least one protein is selected from or derived from a coactivator in the regulation of translation initiation and/or control, nuclear localization proteins, ER localization proteins, endosomal escape proteins, post translation modification proteins, ribosome binding proteins, immune stimulation proteins, Golgi apparatus localization proteins, lysosomal localization proteins, mitochondrial localization proteins, and/or proteins for purification or affinity chromatography, or fragments or variant of any of these.
  • the at least one protein is selected from or derived from a coactivator in the regulation of translation initiation and/or control, nuclear localization proteins, ER localization proteins, endosomal escape proteins, post translation modification proteins, ribosome binding proteins, immune stimulation proteins, Golgi apparatus localization proteins, lysosomal localization proteins, mitochondrial localization proteins, and/or proteins for purification or affinity chromatography, or fragments or variant of any of these.
  • Item 32 The RNA conjugate of items 1 to 5, wherein the sealing element comprises at least one targeting moiety.
  • Item 33 The RNA conjugate of item 32, wherein the at least one targeting moiety can bind to a biological target either covalently or non-covalently.
  • Item 34 The RNA conjugate of item 32 or 33, wherein the at least one targeting moiety comprises a carbohydrate, a lipid, a vitamin, a small receptor ligand, a cell surface carbohydrate binding protein or a ligand thereof, a lectin, an r-type lectin, a galectin, a ligand to a cluster of differentiation (CD) antigen, CD30, CD40, a cytokine, a chemokine, a colony stimulating factor, an interferon, a interleukin, a lymphokine, a monokine, or a mutant, derivative and/or combinations of any of these.
  • CD cluster of differentiation
  • Item 35 The RNA conjugate of items 1 to 5, wherein the sealing element comprises at least one polymer.
  • Item 36 The RNA conjugate of item 35, wherein the at least one polymer comprises therapeutic agents, detectable substances, contrast agents, polyethylene glycol (PEG), polypropylene glycol, a cationic polymer, or any synthetic or naturally occurring macromolecule made up of repeating monomeric units.
  • the at least one polymer comprises therapeutic agents, detectable substances, contrast agents, polyethylene glycol (PEG), polypropylene glycol, a cationic polymer, or any synthetic or naturally occurring macromolecule made up of repeating monomeric units.
  • Item 37 The RNA conjugate of any one of the preceding items, wherein the RNA molecule comprises at least one 5’-cap structure, preferably a capO, cap1 , cap2, or an ARCA cap.
  • Item 38 The RNA conjugate of item 37, wherein the 5’-cap structure is selected from a cap1 structure or a modified cap1 structure, preferably a modified cap1 structure without any diol group.
  • Item 39 The RNA conjugate of any one of the preceding items, wherein the RNA molecule comprises at least one 5’-UTR and/or at least one 3’-UTR.
  • Item 40 The RNA conjugate of item 39, wherein the at least one 5’-UTR comprises or consists of a nucleic acid sequence derived from a 5’-UTR of a gene selected from HSD17B4, AIG1 , alpha-globin (HBA1 , HBA2), ASAH1 , ATP5A1 , COX6C, DPYSL2, HHV5, MDR, MP68, NDUFA4, NOSIP, RPL31 , RPL32, RPL35A, SLC7A3, synthetic origin, TUBB4B, UBQLN2, or from a homolog, a fragment or variant of any one of these genes, preferably selected from HSD17B4.
  • Item 41 The RNA conjugate of items 39 or 40, wherein the at least one 5’-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 30-83, or a fragment or a variant of any of these.
  • Item 42 The RNA conjugate of items 39 to 41 , wherein the at least one 3 -UTR comprises or consists of a nucleic acid sequence derived from a 3 -UTR of a gene selected from PSMB3, AES-12S, ALB7, alpha-globin (HBA1 , HBA2), ANXA4, beta-globin (HBB), CASP1 , COX6B1 , FIG4, GH1 , GNAS, NDUFA1 , RPS9, SLC7A3, TUBB4B or from a homolog, a fragment or a variant of any one of these genes, preferably selected from PSMB3.
  • Item 43 The RNA conjugate of items 39 to 42, wherein the at least one 3 -UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 84-141 , or a fragment or a variant of any of these, preferably selected from SEQ ID NO: 85, 131, 133, 135, 137, 139 or 141 , more preferably selected from SEQ ID NO: 85.
  • Item 44 The RNA conjugate of any one of the preceding items, wherein the at least one coding sequence of the RNA molecule of element A is a codon modified coding sequence, preferably wherein codon modified coding sequence is selected from a C maximized coding sequence, a CAI maximized coding sequence, human codon usage adapted coding sequence, a G/C content modified coding sequence, and a G/C optimized coding sequence, or any combination thereof, preferably wherein the at least one codon modified coding sequence is a G/C optimized coding sequence.
  • Item 45 The RNA conjugate of any one of the preceding items, wherein the at least one coding sequence has a G/C content of at least about 55%, 60%, or 65%.
  • Item 46 The RNA conjugate of any one of the preceding items, wherein the at least one coding sequence encodes at least one peptide or protein suitable for use in treatment or prevention of a disease, disorder or condition.
  • Item 47 The RNA conjugate of any one of the preceding items, wherein the at least one peptide or protein is selected or derived from an antibody, an intrabody, a receptor, a receptor agonist, a receptor antagonist, a binding protein, a CRISPR-associated endonuclease, a chimeric antigen receptor (CAR), a chaperone, a transporter protein, an ion channel, a membrane protein, a secreted protein, a transcription factor, a toxin, a cytotoxic peptide, an enzyme, a peptide or protein hormone, a growth factor, a structural protein, a cytoplasmic protein, a cytoskeletal protein, an allergen, a tumor antigen, a proto-oncogene, an oncogene, a tumor suppressor gene, a neoantigen, a mutated antigen, an antigen of a pathogen, or fragments, epitopes, variants, or combinations of any of these.
  • Item 48 The RNA conjugate of any one of the preceding items, wherein the RNA molecule of element A comprises at least one poly(A) sequence, wherein the at least one poly(A) sequence preferably comprises about 30 to about 500 adenosine nucleotides, preferably about 60 to about 250 adenosine nucleotides, more preferably about 60 to about 150 adenosine nucleotides.
  • Item 49 The RNA conjugate of any one of the preceding items, wherein the RNA molecule of element A additionally comprises at least one poly(C) sequence and/or at least one histone stem-loop sequence, preferably at least one histone stem-loop sequence.
  • Item 50 The RNA conjugate of any one of the preceding items, wherein the RNA molecule of element A comprises, preferably in the following order:
  • Item 51 The RNA conjugate of any one of any one of the preceding items, wherein the RNA molecule is selected from an mRNA, a viral RNA or a self-replicating RNA, preferably an mRNA.
  • Item 52 The RNA conjugate of any one of any one of the preceding items, wherein the RNA molecule comprises at least one modified nucleotide, wherein the at least one modified nucleotide is preferably selected from pseudouridine (ip) or N1 -methylpseudouridine (m1 qj).
  • ip pseudouridine
  • m1 qj N1 -methylpseudouridine
  • Item 53 The RNA conjugate of any one of the preceding items, wherein the RNA molecule is an in vitro transcribed RNA, preferably wherein the RNA in vitro transcription has been performed in the presence of a sequence optimized nucleotide mixture.
  • Item 54 The RNA conjugate of any one of the preceding items, wherein the RNA molecule is a purified RNA, preferably wherein the RNA has been purified by RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, cellulose- based purification, or any combination thereof, preferably wherein the RNA has been purified by RP- HPLC and/or TFF and/or AEX and/or oligo(dT).
  • Item 55 The RNA conjugate of any one of the preceding items, wherein the RNA molecule has an integrity of at least about 50%, preferably of at least about 60%, more preferably of at least about 70%, most preferably of at least about 80%.
  • Item 56 The RNA conjugate of items 2 to 55, wherein element B is conjugated to the 5’-terminus of element A via linker element L, or wherein element B is conjugated to the 3’-terminus of element A via a linker element L, or wherein element B is conjugated non-terminally to element A via a linker element L.
  • Item 57 The RNA conjugate of items 2 to 56, wherein element B is conjugated to the 3'-terminus of element A via linker element L.
  • Item 58 The RNA conjugate of items 2 to 57, wherein the linker element L comprises a bond or linkage that conjugates element L directly to element A or B.
  • Item 59 The RNA conjugate of item 58, wherein the bond or linkage comprises a thioether or disulfide bond, a tertiary amine, an oxime linkage, a thiazolidine linkage, a hydrazone linkage, an amide bond, a click chemistry linkage or/and a Diels-Alder reaction linkage.
  • the bond or linkage comprises a thioether or disulfide bond, a tertiary amine, an oxime linkage, a thiazolidine linkage, a hydrazone linkage, an amide bond, a click chemistry linkage or/and a Diels-Alder reaction linkage.
  • Item 60 The RNA conjugate of items 58 or 59, wherein the bond or linkage comprises a tertiary amine or an amide bond.
  • Item 61 The RNA conjugate of any one of the preceding items, wherein element B that has been used to generate the RNA conjugate comprises an amine group.
  • Item 62 The RNA conjugate of any one of the preceding items, wherein element B that has been used to generate the RNA conjugate comprises the element NH2-(M)n-, wherein M is O, NR N , a bond, C1-C10 alkylene, optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl; preferably a bond, substituted
  • G is a nucleobase, hydrogen, halo, hydroxy, thiol, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted amino, azido, optionally substituted C3-C10 (hetero)cycloalkyl, optionally substituted C6-C10 (hetero)aryl;
  • X is O, S, N(R3), or C(R3)2, wherein each R3 is, independently, H, halo, or optionally substituted C1-C10 alkyl;
  • R1 and R2 are each, independently, hydrogen, hydroxyl, or C1-C6 alkoxy, and wherein if G is nucleobase, R1 and R2 are hydrogen or hydroxyl;
  • M is O, NR N , a bond, optionally substituted C1-C10 alkylene, optionally substituted C2-C10 alkenylene, optionally substituted C2-C10 alkynylene, optionally substituted C2-C10 (hetero)cyclylene, optionally substituted C6-C12 (hetero)arylene, optionally substituted diethylen glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted C2-C100 polyethylene glycol, optionally substituted C1-C30 heteroalkylene, optionally substituted C2-C30 heteroalkenylene or optionally substituted C2-C30 heteroalkynylene; wherein R N is H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; n is 0, 1 , 2, 3, 4, or 5;
  • A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position;
  • RNA conjugate of item 63 wherein linker element L comprises the structure of formula I and wherein formula I is specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are hydrogen or hydroxyl, preferably hydrogen; n is 0; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • Item 65 The RNA conjugate of item 63, wherein linker element L comprises the structure of formula I and wherein formula I is specified in that G is a nucleobase, preferably adenine; X is O; R1 and R2 are hydrogen or hydroxyl, preferably hydrogen; M is a bond, substituted C1 alkyl, C2 alkyl, substituted C2 alkyl, substituted C5 alkyl or C6 alkyl; n is 1 ; A indicates element A of the RNA conjugate that is preferably conjugated to element L at that position; B indicates element B of the RNA conjugate that is preferably conjugated to element L at that position.
  • Item 66 The RNA conjugate of items 2 to 65, wherein element A has been conjugated to element B via linker element L by stepwise solid-phase conjugation, synthetic conjugation or post-synthetic conjugation.
  • Item 67 The RNA conjugate of item 66, wherein the post-synthetic conjugation comprises a solid phase conjugation or in solution conjugation, preferably in solution conjugation.
  • Item 68 The RNA conjugate of items 2 to 67, wherein linker element L is produced by oxidizing the 3’ terminus of element A using an oxidant, preferably sodium periodate, resulting in oxidative ring opening of the sugar at the 3'-terminus of element A into a dialdehyde, followed by condensation of the dialdehyde with a nucleophile group provided by element B, preferably an amine group provided by element B.
  • an oxidant preferably sodium periodate
  • Item 69 The RNA conjugate of any one of the preceding items, wherein the RNA conjugate is reduced, preferably wherein the RNA conjugate is reduced with sodium cyanoborohydride.
  • Item 70 The RNA conjugate of any one of the preceding items, wherein the RNA conjugate is characterized by at least one of the following features selected from increased or prolonged half-life, increased resistance to degradation, increased or prolonged stability, increased expression, reduced induction of innate immune response, reduced induction of proinflammatory cytokines and/or an increased translation efficiency when introduced into a population of cells.
  • RNA conjugate of any one of the preceding items wherein the RNA conjugate is a purified RNA conjugate, preferably wherein the RNA conjugate has been purified by RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, affinity chromatography, cellulose-based purification, or any combination thereof, preferably wherein the RNA conjugate has been purified by RP-HPLC and/or TFF and/or AEX and/or oligo(dT).
  • Item 72 The RNA conjugate of any one of the preceding items, wherein the RNA conjugate has been purified using an affinity chromatography selective for element B.
  • Item 73 The RNA conjugate of items 1 to 72, wherein the RNA molecule of element A comprises:
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene
  • sealing element comprises at least one peptide, preferably at least one peptide derived from a Polyadenylate-binding protein-interacting protein, more preferably the peptide comprises SEQ ID NO: 1 or 2; wherein element B is conjugated to element A via a linker element L, preferably wherein element L comprises the structure of formula I and wherein formula I is further specified according to item 64.
  • Item 74 The RNA conjugate of claim 73, wherein the RNA conjugate has an increased resistance to degradation and an increased translation efficiency of a peptide or protein in a cell, preferably a mammalian cell such as a human cell, wherein resistance to degradation and translation efficiency of the peptide or protein is increased when compared to an RNA molecule lacking element B.
  • Item 75 The RNA conjugate of items 1 to 72, wherein the RNA molecule of element A comprises:
  • a 5’-UTR preferably selected or derived from a 5’-UTR of a HSD17B4 gene
  • a 3’-UTR preferably selected or derived from a 3’-UTR of a PSMB3 gene
  • the sealing element comprises at least one nucleic acid, preferably comprising 32 modified adenosines, more preferably 32 phosphorothioate adenosines or 322 -O-methyl-phosphorothioate adenosines; wherein element B is conjugated to element A via a linker element L, preferably wherein element L comprises the structure of formula I and wherein formula I is further specified according to item 65.
  • Item 76 The RNA conjugate of item 75, wherein the RNA conjugate has an increased resistance to degradation and an increased expression of the encoded peptide or protein in a cell, preferably a mammalian cell such as a human cell, wherein resistance to degradation and expression of the peptide or protein is increased when compared to an RNA molecule lacking element B.
  • Item 77 The RNA conjugate of items 1 to 72, wherein the RNA molecule of element A comprises: (a) a 5’-cap structure, preferably a modified cap1 structure;
  • a 5-UTR preferably selected or derived from a 5 -UTR of a HSD17B4 gene
  • a 3-UTR preferably selected or derived from a 3 -UTR of a PSMB3 gene
  • the sealing element comprises at least one modified oligonucleotide, preferably comprising 4 modified adenosines, more preferably 4 PNA-adenosines; wherein element B is conjugated to element A via a linker element L, preferably wherein element L comprises the structure of formula I and wherein formula I is further specified according to item 65.
  • Item 78 The RNA conjugate of item 77, wherein the RNA conjugate has an increased resistance to degradation and an increased expression of the encoded peptide or protein in a cell, preferably a mammalian cell such as a human cell, wherein resistance to degradation and expression of the peptide or protein is increased when compared to an RNA molecule lacking element B.
  • Item 79 The RNA conjugate of items 1 to 72, wherein the RNA molecule of element A comprises:
  • a 5-UTR preferably selected or derived from a 5 -UTR of a HSD17B4 gene
  • a 3-UTR preferably selected or derived from a 3 -UTR of a PSMB3 gene
  • Item 80 The RNA conjugate of item 79, wherein the RNA conjugate has an increased resistance to degradation and an increased expression of the encoded peptide or protein in a cell, preferably a mammalian cell such as a human cell, wherein resistance to degradation and expression of the peptide or protein is increased when compared to an RNA molecule lacking element B.
  • Item 81 A pharmaceutical composition comprising at least one RNA conjugate as defined in any one of the items 1 to 80.
  • Item 82 The pharmaceutical composition of item 81 , wherein the at least one RNA conjugate is formulated in at least one cationic or polycationic compound.
  • Item 83 The pharmaceutical composition of item 82, wherein the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
  • Item 84 The pharmaceutical composition of items 81 to 83, wherein the at least one RNA conjugate is formulated in lipid-based carriers.
  • Item 85 The pharmaceutical composition of item 84, wherein the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polyplexes, and/or nanoliposomes.
  • Item 86 The pharmaceutical composition of item 84 or 85, wherein the lipid-based carriers are lipid nanoparticles, preferably wherein the lipid nanoparticles encapsulate the RNA conjugate.
  • Item 87 The pharmaceutical composition of items 84 to 86, wherein the lipid-based carriers comprise at least one aggregation-reducing lipid, at least one cationic lipid or ionizable lipid, at least one neutral lipid or phospholipid, and at least one steroid or steroid analog.
  • Item 88 The pharmaceutical composition of item 84 to 87, wherein the lipid-based carriers comprise an aggregation reducing lipid selected from a polymer conjugated lipid, preferably selected from a PEG- conjugated lipid or a PEG-free lipid.
  • Item 89 The pharmaceutical composition of item 88, wherein the polymer conjugated lipid is selected or derived from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K or POZ-lipid.
  • Item 90 The pharmaceutical composition of item 88 or 89, wherein the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid.
  • Item 91 The pharmaceutical composition of items 84 to 90, wherein the lipid-based carriers comprise at least one cationic or ionizable lipid selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
  • Item 93 The pharmaceutical composition of items 84 to 92, wherein the lipid-based carriers comprise a cationic lipid selected or derived from SM-102, SS-33/4PE-15, HEXA-C5DE-PipSS, or compound C26.
  • Item 94 The pharmaceutical composition of items 84 to 93, wherein the lipid-based carriers comprise a neutral lipid selected or derived from DSPC, DHPC, or DphyPE.
  • Item 95 The pharmaceutical composition of items 84 to 94, wherein the lipid-based carriers comprise a steroid or steroid analog selected or derived from cholesterol, cholesteryl hemisuccinate (CHEMS), preferably cholesterol.
  • CHEMS cholesteryl hemisuccinate
  • Item 96 The pharmaceutical composition of items 84 to 95, wherein the lipid-based carriers comprise
  • Item 97 The pharmaceutical composition of items 84 to 96, wherein the lipid-based carriers comprise about 20- 60% cationic lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5- 15% aggregation reducing lipid.
  • Item 98 The pharmaceutical composition of items 84 to 97, wherein the wt/wt ratio of lipid to RNA conjugate in the lipid-based carrier is from about 10:1 to about 60:1 , preferably from about 20:1 to about 30:1 .
  • Item 99 The pharmaceutical composition of items 84 to 98, wherein the N/P ratio of the lipid-based carriers encapsulating the RNA conjugate is in a range from about 1 to about 10, preferably in a range from about 5 to about 7.
  • Item 100 The pharmaceutical composition of items 84 to 99, wherein the lipid-based carriers have a Z-average size in a range of about 50nm to about 120nm.
  • Item 101 A Kit or kit of parts, comprising at least one RNA conjugate of any one of items 1 to 80, and/or at least one pharmaceutical composition of any one of items 81 to 100, optionally comprising a liquid vehicle for solubilising, and, optionally, technical instructions providing information on administration and dosage of the components.
  • Item 102 The RNA conjugate of items 1 to 80, and/or the pharmaceutical composition of items 81 to 100, and/or the kit or kit of parts of item 101 for use as a medicament.
  • Item 103 The RNA conjugate of items 1 to 80, and/or the pharmaceutical composition of items 81 to 100, and/or the kit or kit of parts of item 101 for use in treating or preventing an infectious disease, a tumour disease, or a genetic disease, disorder or condition.
  • Item 104 A method of treating or preventing a disease, disorder or condition, wherein the method comprises applying or administering to a subject in need thereof the RNA conjugate of items 1 to 80, and/or the pharmaceutical composition of items 81 to 100, and/or the kit or kit of parts of item 101.
  • Item 105 The method of treating or preventing a disease, disorder or condition of item 104, wherein the disease, disorder or condition is an infectious disease, a tumour disease, or a genetic disease, disorder or condition.
  • Item 106 The method of treating or preventing a disorder of item 104 or 105, wherein the subject in need is a mammalian subject, preferably a human subject.
  • Item 107 The method of treating or preventing a disorder of items 104 to 106, wherein the applying or administering is performed via intramuscular injection, intradermal injection, transdermal injection, intradermal injection, intralesional injection, intracranial injection, subcutaneous injection, intracardial injection, intratumoral injection, intravenous injection, or intraocular injection, intrapulmonal inhalation, intraarticular injection, sublingual injection.
  • Item 108 A method of producing an RNA conjugate comprising the steps of a. Providing an RNA molecule (element A) encoding at least one protein or peptide as defined in any one of the items 37 to 55; b. Purifying the RNA molecule by at least one step of purification; c. Oxidizing the purified RNA molecule of step b) with an oxidant, preferably sodium periodate, resulting in oxidative ring opening of the sugar at the 3’-terminus of the RNA into a dialdehyde; d.
  • an oxidant preferably sodium periodate
  • RNA conjugate Condensation of the oxidized RNA molecule with a nucleophile, preferably wherein the nucleophile is provided by an element B, more preferably a sealing element as defined in any one of the items 2 to 36; e.
  • Item 109 The method of producing the RNA conjugate of item 108, wherein the providing step a) comprises a step of RNA in vitro transcription of a DNA template encoding the RNA molecule.
  • Item 110 The method of producing the RNA conjugate of item 108 or 109, wherein the providing step a) comprises a step of co-transcriptional capping using a cap analog, preferably a modified cap1 analogue, more preferably a cap analogue without diol group.
  • Item 111 The method of producing the RNA conjugate of items 108 to 110, additionally comprising a step of enzymatic capping that is preferably carried out after step e).
  • Item 112 The method of producing the RNA conjugate of items 108 to 111 , wherein the at least one step of purification carried out in step b) and/or f) is selected from RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, affinity chromatography, cellulose-based purification, or any combination thereof.
  • Item 113 The method of producing the RNA conjugate of items 108 to 112, wherein the at least one step of purification carried out in step f) is an affinity chromatography selective for element B.
  • Item 114 The method of producing the RNA conjugate of items 108 to 113, wherein the method is for producing an RNA conjugate that is characterized by any one of the features of items 1 to 80.
  • Item 115 The method of producing the RNA conjugate of items 108 to 114, additionally comprising a step of formulating the obtained RNA conjugate preferably into lipid-based carriers.
  • Item 116 Use of a sealing element as defined in any one of items 2 to 36, for increasing or prolonging half-life, increasing resistance to degradation, increasing or prolonged stability, increasing expression, reducing induction of innate immune response, reducing induction of proinflammatory cytokines and/or an increasing translation efficiency of an RNA molecule.
  • Item 117 A method of increasing or prolonging protein expression of an RNA molecule comprising a step of conjugating a sealing element as defined in any one of items 2 to 36 to an RNA molecule to generate an RNA conjugate, and applying or administering the RNA conjugate to a cell or a subject.
  • Item 118 The method of item 117, wherein the RNA conjugate is formulated in lipid-based carriers.
  • the reduced groups (except for group 12) show an increased expression on day 6 compared to the unreduced groups and the control group 2. Further details are provided in Example 3 and in the summary of the Example 3.
  • HSkMC Human Skeletal Muscle Cells
  • HSkMC Human Skeletal Muscle Cells
  • the RNA conjugate showed higher expression compared to the RNA molecule lacking a sealing element (control group 2). Further details are provided in Example 8.
  • Figure 17 shows IFN-a2 levels at 24 h after stimulation of PBMC from two different donors with formulated RNA conjugates listed in Table 12.
  • RNA conjugates sealed with two different TLR7/8 antagonists show decreased IFN-a2 levels compared to control groups of LNP formulated RNA lacking a sealing element (Table 13, Group 2 and 4). Further details are provided in Example 9.
  • GM geometric mean.
  • Figure 19 shows the fraction of INFy positive (top) and TNFa positive (bottom) CD8+ cells after i.m.
  • RNA conjugates Group 3, 4, 5, 6 and 7, further details see Table 13
  • All RNA conjugates show increased fraction of INFy and TNFa positive cells compared to control group 2. Further details are provided in Example 10.
  • the present example provides methods of obtaining the RNA conjugates of the invention as well as methods of generating the composition of the invention comprising RNA conjugates formulated in lipid-based carriers.
  • DNA sequences encoding reporter proteins (GpLuc or PpLuc) or proteins for use in therapy were prepared and used for subsequent RNA in vitro transcription reactions. Some DNA sequences were prepared by modifying the wild type or reference encoding DNA sequences by introducing a G/C optimized cds for stabilization and expression optimization.
  • RNA molecules are provided in Table 1 .
  • Linearized DNA templates were used for DNA dependent RNA in vitro transcription (IVT) using T7 RNA polymerase in the presence of a sequence optimized nucleotide mixture (ATP/GTP/CTP/UTP or, alternatively, with N1- methylpseudouridine ml qi (Example 2.3)) and cap analog: m7G(5)ppp(5)(2’OMeA)pG; TriLink (Cap1) and/or m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG; TriLink (3’OMe-Cap1), indicated in Table 1 , under suitable buffer conditions.
  • the obtained RNA IVT reaction was subjected to purification steps comprising RP-HPLC. The purified RNA was used for the reductive amination of periodate-oxidized RNA molecules, the synthesis of the RNA conjugates.
  • Example 1.3 Synthesis of RNA conjugate comprising elements A, L and B - reductive amination of periodate- oxidized RNA molecules
  • Table 2 Overview of sealing elements (NH2-((M)n-B) used in the Example 1 .3
  • Lipid-based carriers were prepared essentially according to the procedures described in WO2015199952, WO2017004143 and WO2017075531. In short, pumps were used to combine an ethanolic lipid solution with an RNA conjugate aqueous solution with at a ratio of about 1 :5 to 1 :3 (vol/vol) in a T-piece system.
  • Other LNPs used in the working examples were prepared using the NanoAssemblrTM microfluidic system (Precision NanoSystems Inc., Vancouver, BC) according to standard protocols which enables controlled, bottom-up, molecular self-assembly of nanoparticles via custom-engineered microfluidic mixing chips.
  • LNPs were composed of 40.9mol% cholesterol, 10mol% DSPC, 47.4mol% ALC-0315, and 1 ,7mol% ALC-0159 (N:P ratio of 6).
  • Other herein used LNPs were composed of38.5mol% cholesterol, 10mol% DSPC, 50mol% SM-102, and 1 ,5mol% DMG-PEG 2000 (N:P ratio of 6).
  • Other herein used LNPs were composed of 28.5mol% cholesterol, 10mol% DPhyPE, 59mol% C26, and 2.5mol% PMOZ4 (N:P ratio of 14).
  • RNA conjugate listed in Table 2, Table 3 and Table 4. Therefore, GpLuc levels were measured in different cell lines for 6 days in comparison to non-transfected cells (Control group 1) and control RNA molecule without sealing element (Control group 2).
  • GpLuc expression was measured in cell supernatants. Supernatants were harvested at each time point of collection and stored at -80°C. 200pl of fresh cell culture medium were added to the cells. For measurement, 10pl were transferred into white LIA assay plates (Greiner Cat. 655075). Plates were introduced into a Hidex Chameleon plate reader with injection device for Renilla-juice containing substrate for Renilla luciferase. Per well, 10OpI of beetle-juice were added and GpLuc lumincescence was measured by Hidex Chameleon plate reader.
  • RNA and RNA conjugates used in the experiment were prepared according to Example 1 and listed in Table 3. Methods used herein are further described in Example 2.
  • RNA conjugates listed in Table 3
  • Table 3 After transfection of RNA conjugates (listed in Table 3) in HeLa cells, increased expression levels were detected compared to the control groups ( Figure 1). All RNA conjugates showed a significant higher long-term expression over 6 days compared to the control groups ( Figure 1).
  • RNAs and RNA conjugates used in the experiment were prepared according to Example 1 , methods used herein are further described in Example 2.
  • Example 2.3 Long term in vitro expression of RNA conjugates with UTP modified RNA molecule in A549 cells
  • RNAs and RNA conjugates used in the experiment were prepared according to Example 1 , methods used herein are further described in Example 2.
  • RNA conjugates with unmodified RNA led to an effective production of the reporter protein in cells. Additionally, the observed expression levels were increased and more stable over 6 days compared to the control group. This effect was shown for RNA conjugates with unmodified RNA (element A) and RNA conjugates with 1 MipU modified RNA (element A). The results demonstrate that the herein tested RNA conjugates substantially increased expression levels and/or long-term expression of the encoded protein which shows the potential of such RNA conjugate for various medical applications.
  • Example 3 Comparison of reduced and unreduced RNA conjugates
  • the goal of the experiment was to investigate the influence of the reduction step with NaBH 3 CN during synthesis of the RNA conjugate (Example 1 .3).
  • GpLuc levels of reduced and unreduced RNA conjugates were measured in A549 cells transfected with RNA conjugates for 6 days in comparison to non-transfected cells (Control group 1) and control RNA molecule without sealing (Control group 2).
  • RNAs and RNA conjugates used in the experiment were prepared according to Example 1 , methods used herein are further described in Example 2.
  • RNA conjugates reduced and unreduced, led to an effective production of the reporter protein in cells. Additionally, the observed expression levels were increased and more stable over 6 days compared to the control group. The reduction step with NaBH3CN showed nearly in all RNA conjugates an additional benefit for high long-term expression.
  • RNA conjugates comprising a sealing element selected from peptides derived from a Polyadenylate-binding protein-interacting protein (PAIP), in particular PAIP1 or PAIP2.
  • PAIP Polyadenylate-binding protein-interacting protein
  • Example 4.1 Long term in vitro expression of RNA conjugates with peptides derived from the PAIR protein in A549 cell line
  • GpLuc levels of RNA conjugates comprising a sealing element selected from PAIP-peptides were measured in A549 cells for 6 days in comparison to non-transfected cells (Control group 1) and control RNA lacking the conjugated peptides derived from PAIP proteins (Control group 2).
  • RNA conjugates comprising a sealing element selected from PAIP-peptides showed a stabilized and increased longterm expression (Figure 6) compared to the control group lacking the conjugated peptides.
  • Example 4.2 Long term in vitro expression in human PBMCs of RNA conjugates with peptides derived from the PAIP protein
  • PBMCs of 4 different donors were isolated according to standard protocols. Cells were seeded into each well of a 96- well plate. PBMC were incubated overnight with 100ng well of RNA conjugates (Table 7, Group 1-4) and control group without the conjugated PAIP peptides in a total volume of 200pl in a humidified 5% CO2 atmosphere at 37°C. 24 hours after transfection, GpLuc levels were measured according to Example 2.
  • RNA conjugates sealed with PAIP-peptides led to a strong expression of the reporter protein (Figure 7) compared to the control group lacking the conjugated peptides derived from a Polyadenylate-binding protein-interacting protein (PAIP), in particular from PAIP1 or PAIP2.
  • PAIP Polyadenylate-binding protein-interacting protein
  • RNA conjugates comprising a sealing element selected from PAIP-peptides shown a beneficial effect on the longterm expression. This leads to the conclusion that there is a positive effect on the expression of the encoded protein and/or the stability of the RNA conjugates.
  • RNA conjugates in human myoblasts. All currently available RNA vaccines are injected intramuscular and prolongated long-term expression in myoblasts could lead to a significant improvement for vaccines.
  • RNA conjugates and control group lacking a sealing element were transfected into HSkMC (Human Skeletal Muscle Cells) cells in two different doses (50ng and 500ng) and the produced protein levels were analyzed over a time period of 5 days.
  • RNAs and RNA conjugates used in the experiment were prepared according to Example 1 and listed in Table 8.
  • HSkMC Human myoblasts
  • HSkMC Human myoblasts
  • Differentiation Medium containing 2% horse serum (Gibco) to induce differentiation.
  • Cells were maintained at 37°C, 5% CO 2 .
  • RNA and RNA conjugates were complexed with Lipofectamine 3000 at a ratio of 1/2.5 (w/v) for 20 minutes in Opti-MEM.
  • RNA conjugates in human myoblast emphasizes the potential of such RNA conjugate for various medical applications such as, in particular, vaccines.
  • Example 6 Long term in vivo expression and reduced inflammatory cytokine levels of RNA conjugates after i.m. and i.v. administration of RNA conjugate
  • RNA conjugates in vivo.
  • effects of RNA administration like induction of inflammatory cytokine levels were measured.
  • mice 5pg (intravenous) or 1 pg (intramuscular) LNP formulated RNA conjugates and control group with formulated RNA lacking a sealing element were delivered by intravenous injection or intramuscular injection.
  • PBS was administered as a second control composition.
  • Luminescence data was collected using standard procedures at 10h, 24h and 48h. IFNa levels were measured by an IFNa ELISA as commonly known in the art. IFNa was measured from serum sample collected at 10h time point.
  • RNAs and RNA conjugates used in the experiment were prepared and formulated in LNPs according to Example 1 and listed in Table 9.
  • Example 6.1 Long term in vivo expression and reduced inflammatory cytokine levels of RNA conjugates after i.m. administration of RNA conjugate
  • RNA conjugates are characterized by equal (Group 3, 5, 6) or even reduced (Group 4) immunogenicity compared to the control group.
  • Example 6.2 Long term in vivo expression and reduced inflammatory cytokine levels of RNA conjugates after i.v. administration of RNA conjugate
  • RNA conjugates shown after 48h a more stable long-term expression of the reporter protein after i.v. administration ( Figure 12).
  • Figure 13 demonstrates that three of four RNA conjugates are characterized by reduced immunogenicity compared to the control group 2.
  • RNA conjugate led to strong long-term in vivo expression after i.m. and i.v. administration.
  • Long-term expression after i.m. or i.v. administration is an important feature of the RNA conjugate and is advantageous in the context of various medical applications. In the medical context of a vaccine this could lead to higher antigen-specific immune responses. In the context medical context of an RNA therapy for long term protein replacement therapy this could lead to reduced injection time points and beneficial protein levels in the patient.
  • RNA conjugate a low induction of inflammatory cytokines upon in vivo administration, especially after i.v. administration, is a particularly important feature of the RNA conjugate and is advantageous in the context of various medical applications.
  • Example 7 Long term in vitro expression of RNA conjugate encoding HNF4alpha in BHK cells
  • RNA conjugate comprising an RNA molecule encoding hepatocyte nuclear factor 4 alpha/HNF4alpha was used (listed in Table 10).
  • the transcription factor HNF4A is a short-lived protein, which, typically, upon delivery of an mRNA encoding for the wild type protein is barely detected 24h post-transfection.
  • engineered HNF4A RNA by conjugating sealing elements to the RNA according to the invention may advantageously increase the expression, activity and stability over wild type HNF4alpha mRNA.
  • HNF4alpha levels were measured in BHK cells for 3h, 6h, 12h, 24h and 48h after transfection of RNA conjugates in comparison to non-transfected cells (Control group 1) and control RNA molecule lacking a sealing element (Control group 2 (comprising a Cap1) and control group 3 (comprising a 3’Ome-Cap1)).
  • BHK Cells were seeded on 6 well plates (400,000 cells in 10OOpl/well) 24 hours before transfection in a compatible complete cell medium. Cells were maintained at 37°C, 5% CO 2 and medium was replaced prior to transfection by 10OOpI Opti-MEM.
  • RNAs and RNA conjugates were complexed with Lipofectamine 2000 at a ratio of 1/1 .5 (w/v) for 20 minutes in Opti-MEM. Lipocomplexed RNA conjugates and control RNA were then added to cells for transfection with 2pg per well in a total volume of 500p I . The HNF4alpha protein levels were measured by Western Blot analysis.
  • RNAs and RNA conjugates used in the experiment were prepared according to Example 1 and listed in Table 10.
  • RNA conjugates led to a higher long-term expression (12h-48h) of HNF4alpha after cell transfection (Figure 14) compared to the two control groups (Group 2 and 3). Notably, all RNA conjugates (Group 4, 5, 6, 7) led to a HNF4alpha expression already after 48h ( Figure 15).
  • RNA conjugates encoding HNF4alpha show that an improved long-term expression of RNA conjugates encoding HNF4alpha and further emphasizes the potential of RNA conjugates according to the invention for various medical applications.
  • Example 8 In vitro expression in PBMCs of an RNA conjugate comprising a TLR7/8 antagonist Material and methods are comparable to Example 4.2. RNAs and RNA conjugates used in the experiment are prepared according to Example 1 and listed in Table 11 .
  • RNA conjugate comprising the sealing element SM-MePS showed higher GpLuc expression levels in all 4 donors compared to the control groups 24 h after administration ( Figure 16).
  • Figure 16 The RNA conjugate comprising the sealing element SM-MePS showed higher GpLuc expression levels in all 4 donors compared to the control groups 24 h after administration ( Figure 16).
  • Figure 16 Example 9: Reduced innate immune activation of LNP formulated unmodified RNA conjugates in vitro
  • the goal of the experiment was to show reduced induction of inflammatory cytokines after administration of LNP formulated RNA conjugates sealed with two different TLR7/8 antagonists.
  • PBMCs of 2 different donors were isolated according to standard protocols. Cells were seeded into a 96-well plate. PBMC were incubated overnight with 2pg per well of LNP formulated RNA conjugates sealed with two different TLR7/8 antagonists (Table 12, Group 3 and 5) and control groups of LNP formulated RNA lacking a sealing element (Table 12, Group 2 and 4) in a total volume of 200pl in a humidified 5% CO 2 atmosphere at 37°C. Untreated cells were used as an additional control (Table 12, Group 1). 24 hours after transfection, supernatants were harvested, and cytokine levels were measured in supernatants via cytokine bead array (LegendPlex) according to the manufacturer’s protocol.
  • GpLuc expression was measured in supernatants.
  • Supernatants were harvested at 24 hours after transfection and stored at -80°C.
  • supernatants were diluted 1 :10. 20pl of diluted supernatants were transferred into white LIA assay plates (Greiner). Plates were introduced into a Tecan plate reader with injection device for Renilla- juice containing substrate for Renilla luciferase. Per well, 50p I of beetle-juice were added and GpLuc lumincescence was measured by Tecan plate reader.
  • PpLuc expression was measured in lysates.
  • PBMC peripheral blood mononuclear cells
  • lysates were lysed using passive lysis buffer (Biotium) at 24 hours after transfection and the lysates were stored at -80°C.
  • 20pl were transferred into white LIA assay plates (Greiner). Plates were introduced into a Tecan plate reader with injection device for Beetle-juice containing substrate for Firefly luciferase. Per well, 50pl of beetle-juice were added and PpLuc lumincescence was measured by Tecan plate reader.
  • RNAs and RNA conjugates used in the experiment are prepared according to Example 1 and listed in Table 12.
  • Example 10 Vaccination with RNA conjugates encoding rabies virus antigen
  • RNA conjugates encoding a rabies virus antigen induce neutralizing antibodies (VNTs) and T-cell responses.
  • 0.1 pg ml qj modified RNA conjugates and control group 1 (unsealed RNA) were injected intramuscularly into mice at day 0 and day 21 . Blood samples were taken on days 21 and 28. Virus neutralization titer (VNT) was analysed each time point. Experimental results are shown in Figure 18 for day 21 and day 28. Splenocytes were harvested on day 28 and analysed. Experimental results from intracellular cytokine staining (ICS) are shown in Figure 19.
  • RNAs and RNA conjugates used in the experiment are prepared according to Example 1 and listed in Table 13.
  • virus neutralization titer According to WHO standards, an antibody titer is considered protective if the respective VNT is at least 0.5IU/ml. Therefore, blood samples were taken from vaccinated mice on day 21 and on day 28 and sera were prepared. These sera were used in fluorescent antibody virus neutralisation (FAVN) test using the cell culture adapted challenge virus strain (CVS) of rabies virus as recommended by the OIE (World Organisation for Animal Health) and first described in Cliquet F., Aubert M. & Sagne L. (1998); J. Immunol.
  • FAVN fluorescent antibody virus neutralisation
  • splenocytes were frozen in FBS/10%DMSO. After thawing, splenocytes were seeded into 96-well plates (2x10 6 cells/well) and stimulated with 1 pg/ml the RAV-G peptide library (JPT) and 2.5pg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37°C in the presence (5h of the 6h) of the mixture of GolgiPlugTM/GolgiStopTM (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells were transferred into medium and kept overnight at 4°C.
  • JPT RAV-G peptide library
  • an anti-CD28 antibody BD Biosciences

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Abstract

La présente invention concerne notamment un conjugué d'ARN qui comprend au moins un élément A et au moins un élément B, l'élément A comprenant ou consistant en une molécule d'ARN contenant au moins une séquence codante codant pour au moins un peptide ou une protéine, et l'élément B comprenant ou consistant en au moins un élément de scellement (élément C). L'élément B peut être conjugué à l'élément A par l'intermédiaire d'un élément de liaison L. Le conjugué d'ARN est avantageusement caractérisé par une résistance accrue à la dégradation dans une cellule et/ou une expression prolongée de la protéine ou du peptide codé dans une cellule. D'autres aspects concernent, entre autres, des procédés de production des conjugués d'ARN, des procédés d'augmentation de l'expression d'un ARN et des utilisations médicales.
PCT/EP2024/073042 2023-08-16 2024-08-16 Conjugués d'arn Pending WO2025036992A1 (fr)

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