RNA combination and composition in which immunostimulatory properties are reduced

Abstract

To provide an RNA combination and a composition in which immunostimulatory properties are reduced.SOLUTION: A combination includes (i) a first component including at least one RNA for treatment, and (ii) a second component including at least one antagonist of at least one RNA sensing pattern recognition receptor. Furthermore, provided is a composition including at least one antagonist of at least one RNA for treatment and at least one RNA sensing pattern recognition receptor. A combination of the two components reduces immunostimulatory properties of the first component, and can promote expression after administration. Furthermore, provided are first and second medical uses, and a method for treating or preventing a disease, a disorder or a condition .SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Introduction] The present invention relates, inter alia, to a combination comprising (i) a first component comprising at least one therapeutic RNA and (ii) a second component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor. Further provided is a composition comprising at least one therapeutic RNA and at least one antagonist of at least one RNA-sensing pattern recognition receptor. Further provided are a first and a second medical use, as well as a method of treating or preventing a disease, disorder or condition.

[0002] RNA-based therapeutics can be used, for example, in passive and active immunotherapy, protein replacement therapy, or genetic engineering. Thus, therapeutic RNAs have the potential to provide highly specific and individualized treatment options for the treatment of a wide variety of diseases, disorders, or conditions.

[0003] In addition to being used as a vaccine, RNA molecules can also be used as therapeutic agents for replacement therapy, for example, for protein replacement therapy in a patient to replace a deleted or mutant protein (e.g., a growth factor or an enzyme). However, the success of developing a safe and effective RNA-based replacement therapy is based on different preconditions compared to vaccines. When encoding an RNA for protein replacement therapy, the therapeutic coding RNA should confer sufficient expression of the protein of interest from the perspective of the expression level and duration of the innate immune system and minimal stimulation in order to avoid inflammation in the treated patient, and should avoid a specific immune response to the administered RNA molecule and the encoded protein.

[0004] The inherent immunostimulatory properties of therapeutic RNAs are considered desirable for vaccines, but this effect can cause unwanted complications in replacement therapies. This is especially true for the treatment of chronic diseases where RNA therapeutics need to be repeatedly administered over long periods of time. The potential ability of therapeutic RNAs to induce innate immune responses can represent a limitation to their in vivo application.

[0005] Induction and / or enhancement of immune responses of the innate and / or adaptive immune system play important roles in a number of diseases. Several innate immune receptors have been identified that are specialized for detecting foreign or damage-associated nucleic acids. One of these groups of nucleic acid-sensing immune receptors is the Toll-like receptor (TLR), a pattern recognition receptor (PRR) that is preferentially located in the endosomal compartments of different immune cell subsets and certain somatic cells. The latter receptors serve to identify pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). The PPRs act as a primary defense against pathogens and control the activation and progression of adaptive immunity not only by inducing inflammatory cytokines, chemokines and interferons, but also by activating B cells and T cells. Among the PPRs, the Toll-like receptors (TLRs) are of particular interest. Their discovery over 30 years ago improved our understanding of the regulation of innate immunity, inflammation and cytokine induction. Stimulation of nucleic acid-sensing receptors typically induces cytokines (e.g., type I interferons) and chemokines to alert neighboring cells and, for example, mobilize immune cells. For example, TLR3, TLR7, TLR8 and TLR9 are intracellular TLRs that recognize nucleic acids (e.g., in the case of RNA) that are taken up by cells via endocytosis and transferred to endosomes. Additional nucleic acid-sensing immune receptors include helicases of the RIG-I family (e.g., RIG-I, MDA5, LGP2), NOD-like receptors, PKR, OAS, SAMHD1, ADAR1, IFIT1 and / or IFIT5.

[0006] Therefore, the induction of innate immune responses mediated mainly by RNA-sensing pattern recognition receptors such as toll-like receptors 7 and 8 impairs the effectiveness of RNA-based therapeutics and thus may lead to a decrease in therapeutic efficacy. Even if the induction of a specific cytokine profile may be advantageous for prophylactic vaccines, for example, the reactogenicity against RNA vaccines characterized by heat and disease must be avoided. Therefore, finding the balance of the induction of innate immune responses to support the adaptive immune response while avoiding fever and disease is an issue in this field.

[0007] In the art, this problem has been partially addressed by using modified RNA nucleotides. By introducing modified nucleotides, therapeutic RNAs can show a decrease in innate immune activation in vivo. However, the modifications also interfere with the recruitment of beneficial RNA-binding proteins and thus can interfere with the activity of therapeutic RNAs, such as protein translation. Therefore, therapeutic RNAs containing modified nucleotides often show a decrease in expression or activity in vivo.

[0008] The prior art describes the use of immunomodulatory oligonucleotides (IROs) with modified CpG motifs as antagonists of TLRs to inhibit and / or suppress TLR-mediated immune responses induced by endogenous and / or exogenous nucleic acids such as modified messenger RNA (mmRNA) therapeutics or DNA used in gene therapy (WO2017136399). Small synthetic oligodeoxynucleotides (ODNs) containing unmethylated deoxycytidine-deoxyguanosine (CpG) dinucleotides can mimic the immune-activating activity of bacterial DNA through recognition by TLR9 (Pohar et al., Selectivity of Human TLR9 for Double CpG Motifs and Implications for the Recognition of Genomic DNA, J Immunol March 1, 2017, 198 (5) 2093-2104 and El-Zayat et al., Toll-like receptors activation, signaling, and targeting: an overview, Bulletin of the National Research Centre(2019) 43:187).

[0009] In summary, it is a problem to reduce the immunostimulatory properties of therapeutic RNA while simultaneously maintaining its efficacy, e.g., its ability to be translated intracellularly and / or induce an adaptive immune response. However, in most therapeutic settings, both characteristics (reduced or low immunostimulatory properties, high translation rate in vivo) are most important for RNA drugs.

[0010] The objectives outlined above are solved by the claimed subject matter of the present invention.

[0011] [Definitions] For clarity and ease of reading, the following definitions are provided. Any technical features referred to for these definitions may be read for each and every embodiment of the present invention.

[0012] Percentages in the context of numbers should be understood as relative to the total number of each item. In other cases, depending on the context, percentages should be understood as weight percentages (wt%).

[0013] About: It is used when the parameter or value does not necessarily have to be identical, i.e., 100% identical. Thus, "about" means that the parameter or value can differ by 0.1% to 20%, preferably 0.1% to 10%, particularly 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. One of ordinary skill in the art knows, for example, that a particular parameter or value can vary slightly based on how that parameter was determined. For example, if defined herein as having a length of "about 1000 nucleotides", the length can differ by 0.1% to 20%, preferably 0.1% to 10%, particularly 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Thus, one of ordinary skill in the art would know in that specific example that the length can branch by 1 to 200 nucleotides, preferably 1 to 100 nucleotides; particularly 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides.

[0014] Adaptive immune response: As used herein, the term "adaptive immune response" is recognized and understood by one of ordinary skill in the art and is intended to refer to, for example, an antigen-specific reaction of the immune system (adaptive immune system). Antigen specificity enables the generation of a reaction tailored to a specific pathogen or pathogen-infected cell. The ability to initiate these tailored reactions is usually maintained in the body by "memory cells" (B cells). In the context of the present invention, the antigen can be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0015] Antibody, antibody fragment: As used herein, the term "antibody" includes both intact antibodies and antibody fragments. Typically, an intact "antibody" is an immunoglobulin that specifically binds to a particular antigen. Antibodies can be members of any immunoglobulin class, including human classes: IgG, IgM, IgE, IgA, and IgD. Typically, an intact antibody is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, and each pair has a "light" chain and a "heavy" chain. An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable portion of the antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments consisting of the heavy and light chain variable regions, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked together by a peptide linker ("ScFv protein"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable regions. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab’ fragments, F(ab’)2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, fragment Fd’, Fd fragments, and isolated complementarity determining regions (CDRs). Suitable antibodies that can be encoded by the therapeutic RNAs of the present invention include monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, Fab fragments, or bispecific antibodies. In the context of the present invention, an antibody can be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0016] Agonist: The term "agonist" is used for a substance that binds to a receptor on a cell and induces a response. Agonists often mimic the action of naturally occurring substances (e.g., ligands).

[0017] Antagonist: The term "antagonist" generally means a substance that attenuates the effect of an agonist.

[0018] Antigen: As used herein, the term "antigen" is recognized and understood by those of ordinary skill in the art, can be recognized by the immune system, preferably the adaptive immune system, and is intended to refer, for example, to a substance that can induce an antigen-specific immune response, such as by the production of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC. Also, fragments, variants, and derivatives of peptides or proteins, such as those derived from cancer antigens, that contain at least one epitope can also be understood as antigens. In the context of the present invention, an antigen can be a translation product of a therapeutic RNA provided (e.g., coding RNA, replicon RNA, mRNA). The term "antigenic peptide or protein" is recognized and understood by those of ordinary skill in the art and is intended to refer, for example, to a peptide or protein derived from a (antigenic) protein that can stimulate the body's adaptive immune system to provide an adaptive immune response. Thus, an "antigenic peptide or protein" contains at least one epitope or antigen (e.g., tumor antigen, viral antigen, bacterial antigen, protozoan antigen) of the protein from which it is derived. In the context of the present invention, an antigen can be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0019] Carrier: The term "carrier" includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent depend on the route of administration of a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in Remington Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co, Easton, PA, 1990.

[0020] Cationic, cationizable: Unless another meaning is clear from the specific context, the term "cationic" means that each structure has a positive charge either permanently or not permanently, but in response to a specific condition such as pH. Thus, the term "cationic" encompasses both "permanently cationic" and "cationizable". As used herein, the term "cationizable" means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH in its surroundings. Also, in a non-aqueous environment where the pH value cannot be measured, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or low activity of hydrogen ions. Whether it is charged or uncharged at that pH or hydrogen ion concentration depends on the individual characteristics of the cationizable or polycationizable compound, particularly the pKa of each cationizable group or atom. In a diluted aqueous environment, the proportion of a cationizable compound, group, or atom having a positive charge can be estimated using the well-known so-called Henderson-Hasselbalch equation by those skilled in the art. For example, if a compound or moiety is cationizable, it is preferably positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, more preferably 6 to 8, even more preferably 9 or less, 8 or less, or 7 or less, most preferably at a physiological pH value, for example about 7.3 to 7.4, i.e., under physiological conditions, particularly under the physiological salt conditions of cells in vivo. In an embodiment, the cationizable compound or moiety is predominantly electrically neutral at a physiological pH value (e.g., about 7.0 to 7.4), but is preferably positively charged at a lower pH value. In some embodiments, the preferred range of pKa for a cationizable compound or moiety is from about 5 to about 7.

[0021] Derived from: The term "derived from", when used throughout this specification in the context of nucleic acids, i.e., for a nucleic acid "derived from" (another) nucleic acid, means that the nucleic acid derived from (another) nucleic acid shares, for example, at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity with the nucleic acid from which it is derived. One skilled in the art knows that sequence identity is typically calculated for nucleic acids of the same type, i.e., for DNA sequences or for RNA sequences. Thus, when DNA is "derived from" RNA, or RNA is "derived from" DNA, in the first step, the RNA sequence is converted to the corresponding DNA sequence (in particular, by substituting U with T throughout the sequence), or vice versa, the DNA sequence is converted to the corresponding RNA sequence (in particular, by substituting T with U throughout the sequence). Thereafter, the sequence identity of the DNA sequence or the RNA sequence is determined. Preferably, a nucleic acid "derived from" a nucleic acid also refers to a nucleic acid modified compared to the nucleic acid from which it is derived, for example, to further increase RNA stability and / or to extend and / or increase protein production. In the context of amino acid sequences, the term "derived from" means that an amino acid sequence derived from (another) amino acid sequence shares, for example, at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity with the amino acid sequence from which it is derived.

[0022] CRISPR-related protein: The terms "CRISPR-associated protein" or "CRISPR-associated endonuclease" are recognized and understood by those skilled in the art. The term "CRISPR-associated protein" refers to an RNA-guided endonuclease that is part of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system (and their homologs, variants, fragments or derivatives), and is used by prokaryotes to confer adaptive immunity against foreign DNA elements. CRISPR-associated proteins include, but are not limited to, Cas9, Cpf1 (Cas12), C2c1, C2c3, C2c2, Cas13, CasX and CasY. As used herein, the term "CRISPR-associated protein" includes wild-type proteins, as well as their homologs, variants, fragments and derivatives. Thus, when referring to an artificial nucleic acid molecule encoding Cas9, Cpf1 (Cas12), C2c1, C2c3, and C2c2, Cas13, CasX, and CasY, the artificial nucleic acid molecule may encode the respective wild-type protein, or its homolog, variant, fragment, and derivative. In addition to Cas9 and Cas12 (Cpf1), there are several other CRISPR-associated proteins suitable for genetic engineering in the context of the present invention, including Cas13, CasX and CasY. In the context of the present invention, a CRISPR-associated protein may be provided by at least one therapeutic RNA of the combination or composition of the present invention.

[0023] Fragment: As used throughout this specification in the context of a nucleic acid sequence or an amino acid (aa) sequence, the term "fragment" can typically be a shorter portion of, for example, the full-length sequence of 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 full-length sequence. In the context of a protein or peptide, the term "fragment" as used throughout this specification can typically include the sequence of a protein or peptide as defined herein, which, with respect to its amino acid sequence (or the encoded nucleic acid molecule), has its N-terminus and / or C-terminus truncated as compared to the amino acid sequence of the original (native) protein (or the encoded nucleic acid molecule). Thus, such truncation can occur at the aa level or, correspondingly, at the nucleic acid level. Thus, the sequence identity with respect to such fragments as defined herein can preferably refer to the entire protein or peptide as defined herein, or the entire (encoding) nucleic acid molecule of such a protein or peptide. Fragments of antigenic proteins or peptides can contain at least one epitope of these proteins or peptides. Furthermore, protein domains, such as extracellular domains, intracellular domains, or transmembrane domains, and truncated or cleaved forms of proteins can also be understood to include fragments of the protein.

[0024] Heterologous: As used throughout this specification in the context of a nucleic acid sequence or an amino acid sequence, the term "heterologous" or "heterologous sequence" refers to a sequence (e.g., DNA, RNA, amino acid), recognized and understood by one of ordinary skill in the art, and is intended to refer to a sequence derived from a different gene, a sequence derived from a different allele, or a sequence derived from a different species. Two sequences are typically understood to be "heterologous" if they are not derived from the same gene or from the same allele. That is, heterologous sequences can be derived from the same organism, but they are not, in nature, present in the same nucleic acid molecule, for example, as the same RNA or protein.

[0025] (Sequence) identity: As used throughout this specification in the context of nucleic acid or amino acid sequences, the term "identity" is recognized and understood by those skilled in the art and is intended, for example, to refer to the percentage of two sequences that are identical. To determine the percentage of two sequences that are identical, the sequences can be aligned and subsequently compared to each other, for example, for a nucleic acid sequence or an amino acid (aa) sequence as defined herein, preferably an aa sequence encoded by a nucleic acid sequence as defined herein or the aa sequence itself. Thus, for example, a position in the first sequence can be compared to the corresponding position in the second sequence. If the position in the first sequence is occupied by the same residue as the position in the second sequence, the two sequences are identical at this position. If not, the sequences are different at this position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted into the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted into the second sequence to allow for further alignment. In this case, the percentage of the two sequences that are identical is a function of dividing the number of identical positions by the total number of positions including positions occupied only in one of the sequences. The percentage of the two sequences that are identical can be determined using an algorithm, for example, an algorithm incorporated into the BLAST program.

[0026] Immune response: The term "immune response" is recognized and understood by those skilled in the art and is intended, for example, to refer to a specific response of the adaptive immune system to a particular antigen (so-called specific or adaptive immune response), or a non-specific response of the innate immune system (so-called non-specific or innate immune response), or a combination thereof.

[0027] Immune system: The term "immune system" is recognized and understood by those skilled in the art and is intended to refer to, for example, the system of an organism that can protect the organism from infection. When a pathogen passes through the physical barriers of an organism and successfully invades the organism, the innate immune system provides an immediate but non-specific response. If the pathogen evades this innate response, vertebrates have a second defense mechanism, the adaptive immune system. Here, the immune system adapts its response during infection to improve the recognition of the pathogen. Subsequently, this improved response is retained in the form of immunological memory after the pathogen has been eliminated, enabling the adaptive immune system to initiate a faster and more powerful attack each time it encounters this pathogen. According to this, the immune system includes the innate and adaptive immune systems. Each of these two components typically contains so-called humoral and cellular components.

[0028] Treatment: The term "treatment" generally refers to an approach intended to obtain a beneficial or desired result, which may include alleviation of symptoms, or delay or improvement of disease progression.

[0029] Messenger RNA (mRNA): The term "messenger RNA" (mRNA) refers to a type of RNA molecule. In vivo, usually through transcription of DNA, so-called immature RNA is produced, and this RNA undergoes processing to become so-called messenger RNA, which is usually abbreviated as mRNA. Typically, mRNA contains a 5'-cap, 5'-UTR, open reading frame / coding sequence, 3'-UTR, and poly(A).

[0030] Nucleoside: The term "nucleoside" generally refers to a compound consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base.

[0031] Nucleotide: The term "nucleotide" generally refers to a nucleoside containing a phosphate group attached to the sugar.

[0032] Nucleic acid sequence, RNA sequence: The terms "nucleic acid sequence" or "RNA sequence" are recognized and understood by those skilled in the art and are intended to refer, for example, to the specific and individual order of its nucleotides or amino acids, respectively.

[0033] Variant (of a sequence): The term "variant" as used throughout this specification in the context of a nucleic acid sequence is recognized and understood by those skilled in the art and is intended, for example, to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence. For example, 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 be at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence from which it is derived. The variant is preferably a functional variant in the sense that it retains at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence from which it is derived. A "variant" of a nucleic acid sequence may have a nucleotide identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% over a range of at least 10, 20, 30, 50, 75, or 100 nucleotides of such a nucleic acid sequence.

[0034] As used throughout this specification in the context of proteins or peptides, the term "variant" is recognized and understood by those skilled in the art, and is intended to refer to a protein or peptide variant having an amino acid sequence that differs from the original sequence in one or more mutations, such as one or more substituted, inserted and / or deleted amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, such as their specific antigenic properties, compared to the full-length native protein. The "variants" of proteins or peptides as defined herein may include conservative amino acid substitutions (s) compared to their native, i.e., non-mutated physiological sequences. A "variant" of a protein or peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a range of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Preferably, variants of a protein include functional variants of the protein, which means that the variant exhibits the same effect or functionality as the protein from which it is derived, or at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the effect or functionality.

[0035] 〔BRIEF DESCRIPTION OF THE INVENTION〕 The present invention is based on the finding that co-administration of a component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor results in reduced (innate) immune activation induced by therapeutic RNA, for example compared to administration of the corresponding therapeutic RNA alone. Surprisingly, co-administration of a component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor preferably increases and / or prolongs the expression of a peptide or protein encoded by the therapeutic RNA.

[0036] As outlined in the Examples section, the inventors have found that the addition of chemically modified oligonucleotides has an immunosuppressive effect on co-administered immunostimulatory RNA sequences (“RNA adjuvants”) (see, for example, Figure 1A). Furthermore, the inventors have shown that chemically modified oligonucleotides efficiently antagonize the immunostimulatory effects of RNA (see, for example, Example 2 (in vitro) or Example 3 (in vivo)), typically the unwanted side effects induced by RNA-sensing receptors. Oligonucleotides used herein have been described to antagonize Toll-like receptors (TLRs) 7 and 8, RNA-sensing pattern recognition receptors involved in the innate immune response (see Schmitt et al., 2017. RNA 23:1344-135). The present invention is based on the finding that a combination or composition comprising at least one antagonist of at least one RNA-sensing receptor and at least one therapeutic RNA can reduce the immunostimulatory properties of said at least one therapeutic RNA. Unexpectedly, the addition of the antagonistic oligonucleotide increases and / or prolongs the expression of the protein encoded by the co-administered therapeutic RNA, suggesting that a combination or composition comprising an antagonist of at least one RNA-sensing pattern recognition receptor (e.g., a TLR7 antagonist) and a therapeutic RNA (e.g., an mRNA) results in a decrease in immunostimulation and an increase and / or prolongation of protein expression, which is most important for most RNA-based drugs.

[0037] In a first aspect, the invention relates to a combination comprising (i) at least one first component comprising at least one therapeutic RNA, and (ii) at least one second component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor.

[0038] In a second aspect, the invention relates to a pharmaceutical composition comprising (i) at least one therapeutic RNA, preferably as described in the first aspect; (ii) at least one antagonist of at least one RNA-sensing pattern recognition receptor, preferably as described in the first aspect, and optionally at least one pharmaceutically acceptable carrier, or consisting of the foregoing.

[0039] In a third aspect, the invention relates to a kit or kit of parts comprising the first and second components of the combination of the first aspect and / or comprising the composition of the second aspect.

[0040] In a fourth aspect, the invention relates to the combination of the first aspect, the composition of the second aspect, or the kit or kit of parts of the third aspect for use as a medicament.

[0041] In a further aspect, the invention relates to the combination of the first aspect, the composition of the second aspect, or the kit or kit of parts of the third aspect for use in chronic treatment or as a vaccine. Other aspects relate to methods of treating or preventing a disease, disorder or condition, methods of reducing (innate) immunostimulation of a therapeutic RNA, methods of reducing the reactogenicity of a therapeutic RNA composition, and methods of increasing and / or prolonging the expression of a peptide or protein encoded by a (coding) therapeutic RNA.

[0042] 〔DETAILED DESCRIPTION OF THE INVENTION〕 This application is filed as part of the description of this application (WIPO Standard ST.25) together with an electronic sequence listing. The matters contained in the electronic form of the sequence listing filed with this application are hereby incorporated by reference in their entirety into this specification. For many sequences, the sequence listing also provides additional details regarding, for example, specific structural features, sequence modifications, GenBank identifiers, or additional detailed information. In particular, such information is provided under the numerical identifier <223> of the WIPO Standard ST.25 sequence listing. Accordingly, the information provided under the said numerical identifier <223> is hereby expressly included in its entirety in this specification and must be understood as an essential part of the description of the underlying invention.

[0043] [Combination] In a first aspect, inter alia, a combination is targeted that comprises a first component comprising a therapeutic RNA and a second component comprising an antagonist of an RNA-sensing pattern recognition receptor.

[0044] In the context of the present invention, the term "combination" preferably means that at least one therapeutic RNA (referred to herein as the "first component") and at least one antagonist of at least one RNA-sensing pattern recognition receptor (referred to herein as the "second component") occur in combination. Accordingly, the said combination may occur as one composition containing all of these components in one and the same composition or mixture (however, as separate entities), or as a kit of parts, with the different components forming different parts of the kit of parts (as defined in the third aspect). Accordingly, administration of the first and second components of the present combination can be carried out either at the same administration site or at different administration sites, either simultaneously or at an appropriate interval, as further outlined below. The components may be formulated together as a co-formulation (as further described in the context of the second aspect), or may be formulated as different separate formulations (and optionally combined after formulation), as outlined below.

[0045] In a first aspect, the combination comprises (i) at least one first component comprising at least one therapeutic RNA, and (ii) at least one second component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor. The combination comprises.

[0046] Advantageous embodiments and features of the at least one antagonist of the at least one RNA-sensing pattern recognition receptor of the second component are described below. In particular, all of the described embodiments and features of the at least one antagonist described in the context of the combination (first aspect) of the invention are equally applicable to the at least one antagonist of the pharmaceutical composition (second aspect), or to the kit or parts kit (third aspect), or to any further aspect described herein (for example medical use, method of treatment).

[0047] As used throughout this specification, the term "pattern recognition receptor" (PRR) is recognized and understood by those skilled in the art and is intended to refer to receptors that are part of, for example, the innate immune system. Germline-encoded PRRs play a role in sensing the presence of microbial-specific molecules (such as bacterial or viral DNA or RNA) through recognition of conserved structures called pathogen-associated molecular patterns (PAMPs). Recent evidence has shown that PRRs are also responsible for recognizing endogenous molecules released from damaged cells, which are called damage-associated molecular patterns (DAMPs). Currently, four different classes of PRR families have been identified. These families include transmembrane proteins such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), as well as cytoplasmic proteins such as retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs). Based on their localization, PRRs can be divided into membrane-bound PRRs and cytoplasmic PRRs and are expressed not only in macrophages and DCs but also in various non-professional immune cells (Takeuchi and Akira 2010Pattern Recognition Receptors and Inflammation, Cell, Volume 140, ISSUE 6, P805-820).

[0048] Typical pattern recognition receptors (PRRs) in the context of the present invention are Toll-like receptors, NOD-like receptors, RIG-I-like receptors, PKR, OAS1, IFIT1, and IFIT5.

[0049] As used throughout this specification, the term "innate immune system", also known as the non-specific (or unspecific) immune system, is recognized and understood by those of skill in the art and is intended to refer to a system that typically includes cells and mechanisms that defend a host from infection by other organisms in a non-specific manner. This means that while cells of the innate immune system can recognize and respond to pathogens in a general way, unlike the adaptive immune system, they do not confer long-lasting or protective immunity to the host. The innate immune system can be activated, for example, by ligands of "pattern recognition receptors" (PRRs) (e.g., PAMPs), or by other adjuvants such as lipopolysaccharides, TNF-α, CD40 ligand, monokines, IL-1, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-α, IFN-β, IFN-γ, GM-CSF, G-CSF, M-CSF, LT-β, TNF-α, growth factors and hGH, ligands of human Toll-like receptors TLR1, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, ligands of mouse Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, ligands of NOD-like receptors, ligands of RIG-I-like receptors, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibacterial agents, antiviral agents, ligands of PKR and OAS1 (e.g., long double-stranded RNA) or ligands of IFIT1 and IFIT5 (5’ppp RNA).

[0050] Typically, the response of the innate immune system (e.g., after sensing RNA) involves mobilizing immune cells to the site of infection through the production of chemokines, which are special chemical messengers called cytokines; activating the complement cascade; identifying and removing foreign substances present in organs, tissues, blood, and lymph by special white blood cells; activating the adaptive immune system; and / or acting as physical and chemical barriers against infectious agents. Typically, protein synthesis also decreases during the innate immune response. The inflammatory response is regulated by inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6. These cytokines are multifunctional proteins that regulate cell death in inflamed tissues, modify vascular endothelial permeability, mobilize blood cells to inflamed tissues, and induce the production of acute-phase proteins.

[0051] PRRs can be activated by PAMPs derived from viruses, bacteria, fungi, and protozoa, across a wide variety of pathogen-associated molecular patterns (PAMPs), such as lipoproteins, carbohydrates, lipopolysaccharides, and various types of nucleic acids (DNA, RNA, dsRNA, uncapped RNA or 5’ppp RNA). PRRs can be present in various compartments of the cell (e.g., located in the membrane of endosomes or in the cytoplasm). Upon sensing PAMPs, PRRs trigger signaling cascades that lead, inter alia, to the expression of, for example, cytokines, chemokines. For example, toll-like receptor 3 (TLR-3) typically detects long double-stranded RNA (>40 base pairs) and is also expressed on the surface of certain cell types. The expression of TLR7 in the human immune system is typically limited to B cells and pDCs, and TLR8 is preferentially expressed in myeloid immune cells. As a result, TLR7 ligands promote the activation of B cells and the production of large amounts of IFN-α in plasmacytoid dendritic cells (pDCs), while TLR8 induces the secretion of large amounts of IL-12p70 in myeloid immune cells. TLR8 selectively detects ssRNA, while TLR7 mainly detects short stretches of dsRNA but has also been demonstrated in the art to accommodate certain ssRNA oligonucleotides. The TLR9 receptor is mainly expressed in human B cells and plasmacytoid dendritic cells and detects single-stranded DNA containing unmethylated CpG dinucleotides. In addition to cytokine induction, some RNA-sensing pattern recognition receptors of the innate immune system can inhibit protein translation upon binding of their agonists (e.g., dsRNA, 5’ppp RNA), for example. It has been taught that the binding of long double-stranded RNA activates PKR to phosphorylate eIF2a and inhibit mRNA translation. IFIT1 and IFIT5 have been taught to bind to 5’ppp RNA and block eIF2a, thereby inhibiting the translation of mRNA molecules (reviewed in Hartmann, G. ”Nucleic acid immunity” Advances in immunology.Vol.133Academic Press, 2017.121-169).

[0052] Accordingly, as used herein, the term "RNA-sensing pattern recognition receptor" refers to the types of PRRs that can sense RNA. "Sensing" in that context must be understood as the ability of the receptor to bind to RNA and as a result trigger a downstream signaling cascade (such as the induction of cytokines or, for example, the inhibition of translation).

[0053] Accordingly, the term "antagonist of at least one RNA-sensing pattern recognition receptor" relates to a compound that can inhibit and / or suppress a PRR-mediated immune response induced by the therapeutic RNA of the invention. Further, such an antagonist can attenuate the effect (such as a PRR-mediated immune response) of an agonist (such as an immunostimulatory RNA species).

[0054] Accordingly, at least one RNA-sensing pattern recognition receptor preferably induces cytokines upon binding of an RNA agonist. Such an RNA agonist may be single-stranded RNA, double-stranded RNA, or 5'-triphosphate RNA (5'ppp RNA).

[0055] Alternatively or additionally, at least one RNA-sensing pattern recognition receptor may inhibit translation upon binding of an RNA agonist. Such an RNA agonist may be single-stranded, double-stranded, or 5'-triphosphate RNA (5'ppp RNA).

[0056] Advantageously, at least one antagonist of the second component reduces the cytokine induction of at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist and / or reduces the translation inhibition by at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist.

[0057] Thus, in a preferred embodiment, administration of a combination of at least one therapeutic RNA of a first component and at least one antagonist of at least one RNA-sensing pattern recognition receptor of a second component results in a reduction of the innate immune response as compared to administering at least one therapeutic RNA of the first component without combining it with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component.

[0058] Thus, administration of the combination (i.e., administration of the first and second components) to a cell, tissue, or organism results in a reduction of (innate) immune activation as compared to administration of the corresponding first component alone.

[0059] In a further embodiment, administration of the composition (i.e., administration of the first and second components) to a cell, tissue, or organism results in essentially the same or at least equivalent (innate) immune activation as compared to administration of a control RNA having the same RNA sequence and containing modified nucleotides (e.g., as defined herein).

[0060] Induction or activation or stimulation of the innate immune response as described above is typically determined by measuring induction of cytokines.

[0061] Preferably, the reduction of innate immune activation is preferably characterized by a reduced level of at least one cytokine selected from Rantes, MIP-1α, MIP-1β, McP1, TNFα, IFNγ, IFNα, IFNβ, IL-12, IL-6, or IL-8.

[0062] The term "reduced level of at least one cytokine" should be understood to mean that administration of the composition results in a reduction of cytokine induction to a certain percentage as compared to a control (e.g., the first component alone).

[0063] Thus, the reduced innate immune activation in the context of the present invention is preferably characterized by a reduced level of at least one cytokine selected from Rantes, MIP-1α, MIP-1β, McP1, TNFα, IFNγ, IFNα, IFNβ, IL-12, IL-6, or IL-8, wherein the reduced level of the at least one cytokine is a reduction of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Preferably, the reduced level of the at least one cytokine is a reduction of at least 30%.

[0064] Methods for assessing (innate) immune activation (i.e., induction of, for example, Rantes, MIP-1α, MIP-1β, McP1, TNFα, IFNγ, IFNα, IFNβ, IL-12, IL-6, or IL-8) by therapeutic RNA in a particular cell / organ / tissue are well known to those skilled in the art. Typically, the (innate) immune activation of a therapeutic RNA in combination with a second component is compared to the (innate) immune activation of the therapeutic RNA alone (or together with a control RNA containing modified nucleotides) (i.e., without the (additional) administration of the second component). The same conditions (e.g., the same cell line, the same organism, the same application route, the same detection method, the same amount of therapeutic RNA, the same RNA sequence, etc.) should be used (if possible) to enable a valid comparison. Those skilled in the art understand how to make a comparison between the combinations of the present invention and their respective control RNAs (control RNAs having the same RNA sequence, either the therapeutic RNA alone or containing modified nucleotides).

[0065] In the context of the present invention, cytokine induction is measured by administration of the combination to cells, tissues or organisms, preferably hPBMCs, Hela cells or HEK cells. Preferred in that context are hPBMCs. When the combination (or corresponding control) is administered to hPBMCs, Hela cells or HEK cells, an assay is performed to measure cytokine levels. Cytokines secreted into the culture medium or supernatant can be quantified by techniques such as bead-based cytokine assays (e.g., cytometric bead array (CBA)), ELISA, and Western blot.

[0066] Preferably, a bead-based cytokine assay, most preferably a cytometric bead array (CBA), is performed to measure cytokine induction in cells after administration of the combination (and their corresponding controls).

[0067] The CBA can quantify multiple cytokines from the same sample. The CBA system uses flow cytometry and a wide range of fluorescence detection provided by antibody-coated beads to capture cytokines. Each bead in the array has a unique fluorescence intensity so that the beads can be mixed and acquired simultaneously. An appropriate CBA assay in that context is described in the 2012 BD Biosciences Application Note from Reynolds et al., "Quantification of Cytokines Using the BD (Trademark) FACSVerse System and BD (Trademark) Cytometric Bead Array in FCAP Array (Trademark) Software". An exemplary CBA assay for determining cytokine levels is described in the Examples section of the present invention.

[0068] In various embodiments, at least one RNA-sensing pattern recognition receptor is an endosomal receptor or a cytoplasmic receptor. In preferred embodiments, at least one RNA-sensing pattern recognition receptor is an endosomal receptor. A non-limiting list of exemplary endosomal RNA-sensing pattern recognition receptors includes TLR3, TLR7, or TLR8. In that context, "endosomal" must be understood to be localized to the endosome or to the endosomal membrane. A non-limiting list of exemplary cytoplasmic RNA-sensing pattern recognition receptors includes RIG1, MDA5, NLRP3, or NOD2.

[0069] In various embodiments, at least one RNA-sensing pattern recognition receptor is a receptor for single-stranded RNA (ssRNA) and / or a receptor for double-stranded RNA (dsRNA). A non-limiting list of exemplary RNA-sensing pattern recognition receptors for dsRNA includes TLR3, RIG1, MDA5, NLRP3, or NOD2. A non-limiting list of exemplary RNA-sensing pattern recognition receptors for ssRNA includes TRL7, TLR8, RIG1, NLRP3, or NOD2.

[0070] Thus, in a preferred embodiment, at least one second component comprises at least one antagonist of at least one RNA-sensing pattern recognition receptor, and at least one RNA-sensing pattern recognition receptor is selected from Toll-like receptors (TLRs), and / or retinoic acid-inducible gene I-like receptors (RLRs), and / or NOD-like receptors and / or PKR, OAS, SAMHD1, ADAR1, IFIT1 and / or IFIT5.

[0071] In a preferred embodiment, at least one second component comprises at least one antagonist of at least one RNA-sensing pattern recognition receptor, where at least one RNA-sensing pattern recognition receptor is selected from PKR, OAS, SAMHD1, ADAR1, IFIT1 and / or IFIT5.

[0072] In a preferred embodiment, at least one Toll-like receptor is selected from TLR3, TLR7, TLR8 and / or TLR9. In a particularly preferred embodiment, the Toll-like receptor is selected from TLR7 and / or TLR8. Thus, in the context of the present invention, "at least one antagonist of at least one RNA-sensing pattern recognition receptor" is preferably an antagonist of a Toll-like receptor selected from TLR3, TLR7, TLR8 and / or TLR9, preferably TLR7 and / or TLR8.

[0073] In a preferred embodiment, at least one retinoic acid-inducible gene-I-like receptor (RLR) is selected from RIG-1, MDA5, LGP2, cGAS, AIM2, NLRP3, and / or NOD2. In a particularly preferred embodiment, the RLR is RIG-1 and / or MDA5. Thus, "at least one antagonist of at least one RNA-sensing pattern recognition receptor" is preferably an antagonist of a retinoic acid-inducible gene-I-like receptor (RLR) selected from RIG-1, MDA5, LGP2, cGAS, AIM2, NLRP3, and / or NOD2, preferably RIG-1, MDA5.

[0074] In the context of the present invention, at least one antagonist of the second component as defined herein may be selected from nucleotides, nucleotide analogs, nucleic acids, peptides, proteins, antibodies, small molecules, lipids, or fragments, variants, or derivatives of any of these.

[0075] In some embodiments, the antagonist is a substituted quinoline compound, a substituted quinazoline compound, a tricyclic TLR inhibitor (e.g., mianserin, desipramine, cyclobenzaprine, imipramine, ketotifen, and amitriptyline), the vaccinia virus A52R protein (US 20050244430), Polymyxin-B (a specific inhibitor of LPS bioactivity), BX795, chloroquine, hydroxychloroquine, CU-CPT8m, CU-CPT9a, CU-CPT9b, CU-CPT9c, CU-CPT9d, CU-CPT9e, CU-CPT9f, CLI-095, RDP58, ST2825, ML120B, PHA-408, insulin (clinical trial NCT01151605), an oligodeoxynucleotide (ODN) that suppresses the CpG-induced immune response, a G-rich ODN, and a TLR antagonist comprising an ODN having a TTAGG motif. In some embodiments, the TLR antagonist includes those described in patent or patent application US20050119273, WO2014052931, WO2014108529, US20140094504, US20120083473, US8729088, and US20090215908.In some embodiments, the TLR inhibitor includes the following: ST2 antibody; sST2-Fc (functional murine soluble ST2-human IgG1 Fc fusion protein; see Biochemical and Biophysical Research Communications, December 29, 2006, Vol. 351, no. 4, 940-946); CRX-526 (Corixa); lipid IVA; RSLA (Rhodobacter sphaeroides lipid A); E5531 ((6-0-{2-deoxy-6-0-methyl-4-0-phosphono-3-0-[(R)-3-Z-dodec-5-enoyloxydodecyl]-2-[3-oxo-tetradecanoylamino]--0-phosphono-a-D-glucopyranose tetrasodium salt); E5564 (a-D-glucopyranose, 3-0-decyl-2-deoxy-6-0-[2-deoxy-3-0-[(3R)-3-methoxydodecyl]-6-0-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-0-phosphono--D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-l-(dihydrogen phosphate), tetrasodium salt); compound 4a (hydrocinnamoyl-l-valylpyrrolidine; see PNAS, June 24, 2003, vol. 100, no. 13, 7971-7976); CPG 52364 (Coley Pharmaceutical Group); LY294002 (2-(4-morpholinyl)-8-phenyl 4H-1-benzopyran-4-one); PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one); chloroquine; C2 dimer with propylene spacer as an antagonist of TLR7 / 8 (see Table A) and immunomodulatory oligonucleotides (see US Patent Application Publication No. 2008 / 0089883). Further suitable TLR antagonists are described by Patinote et al. (Patinote et al., Agonist and toll-like receptor 7 and 8 antagonist ligands: Ingenious tools for therapeutic purposes, Eur J Med Chem. May 1, 2020; 193: 112238).

[0076] Accordingly, suitable chemical compounds, for example, small molecule compounds that can be used as antagonists in the context of the present invention, are selected from the following: chloroquine, CU-CPT9a, hydroxychloroquine, quinacrine, monensin, bafilomycin A1, wortmannin, β-aminoarteether maleate, (+)-morphinan, 9-aminoacridine, 4-aminoquinoline, 7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolin-1-ylamine; 1-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]quinolin-4-ylamine; 1,6-dimethyl-2,3-dihydro-1H-pyrrolo[2,3-b]quinolin-4-ylamine; 6-bromo-1-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]quinolin-4-ylamine; 1-methyl-2,4,5-tetrahydro-1H-azepino[2,3-b]quinolin-6-ylamine; 3,3-dimethyl-3,4-dihydro-acridin-9-ylamine; 1-benzyl-2,3-dihydro-1H-pyrrolo[2,3b]quinolin-4-ylamine; 6-methyl-1-phenyl-2,3-dihydro-1H-pyrrolo[2,3-b]quinolin-4-ylamine; N*2*,N*2*-dimethyl-quinoline-2,4-diamine, 2,7-dimethyl-dibenzo[b,g][1,8]naphthyridin-11-ylamine; 2,4-dimethyl-benzo[b][1,8]naphthyridin-5-ylamine; 7-fluoro-2,4-dimethyl-benzo[b][1,8]naphthyridin-5-ylamine; 1,2,3,4-tetrahydro-acridin-9-ylamine Tacrine hydrochloride hydrate; 2,3-dihydro-1H-cyclopenta[b]quinolin-9-ylamine; 2,4,9-trimethyl-benzo[b][1,8]naphthyridin-5-ylamine; 9-amino-3,3-dimethyl-1,2,3,4-tetrahydro-acridin-1-ol and 7-ethoxy-N*3*-furan-2-ylmethyl-acridine-3,9-diamine; quinazoline, N,N-dimethyl-N’-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-3,4-dihydro-quinazolin-4-yl}-ethane-1,2-diamine;N’-[6,7-Dimethoxy-2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-N,N-dimethyl-ethane-1,2-diamine; N’-[6,7-Dimethoxy-2-(4-methyl-piperazin-1-yl)-quinazolin-4-yl]-N,N-dimethyl-ethane-1,2-diamine; N,N-Dimethyl-N’-(2-phenyl-quinazolin-4-yl)-ethane-1,2-diamine; Dimethyl-(2{2-[4-(4-methyl-piperazin1-yl)-phenyl}-quinazolin-4-yloxy}-ethyl)-amine; N’-(2-biphenyl-4-yl-quinazolin-4-yl)-N,N-dimethyl-ethane-1,2-diamine, and, Dimethyl-[2-(2-phenyl-quinazolin-4-yloxy)-ethyl]-amine, statin, atorvastatin.;

[0077] In some embodiments, suitable chemical compounds for use as, for example, small molecule compounds, are selected from: chloroquine (C 18 H 26 ClN3), an anti-malarial drug having anti-inflammatory, and potential chemical and radiation sensitizing activities, or CU-CPT9A (C 17 H 15 NO2), a potent and selective inhibitor of Toll-like receptor 8 (see Table A) (Zhang, S. et al, 2018. Small-molecule inhibition of TLR8 through stabilization of its resting state. Nat Chem Biol, 14(1): 58-64 and Mohamed et al, effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma, Liver International (2015)).

[0078]

Table 1

[0079] In a preferred embodiment, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a nucleic acid.

[0080] The terms "nucleic acid" or "nucleic acid molecule" are recognized and understood by those skilled in the art and are intended to refer to a molecule, preferably comprising or consisting of nucleic acid components. The term nucleic acid molecule preferably refers to DNA and RNA or mixtures thereof. It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers (natural and / or modified) covalently linked to each other by phosphodiester bonds of a sugar / phosphate backbone. Examples of suitable modified nucleotides are LNA or PNA nucleotides. The term "nucleic acid" also encompasses modified nucleic acid molecules such as base-modified, sugar-modified or backbone-modified DNA or RNA molecules as defined herein. The term "nucleic acid" also encompasses single-stranded, double-stranded, and branched nucleic acid molecules.

[0081] In a particularly preferred embodiment, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a single-stranded nucleic acid, for example for single-stranded RNA.

[0082] In an alternative embodiment, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a double-stranded nucleic acid, for example for double-stranded RNA.

[0083] In a preferred embodiment, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a nucleic acid comprising or consisting of nucleotides selected from DNA nucleotides, RNA nucleotides, PNA nucleotides, and / or LNA nucleotides, or analogs or derivatives of any of these.

[0084] In particularly preferred specific examples, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a single-stranded nucleic acid, where the nucleic acid comprises or consists of nucleotides selected from DNA nucleotides, RNA nucleotides, PNA nucleotides, and / or LNA nucleotides, or analogs of any of these.

[0085] In other embodiments, the "at least one antagonist of at least one RNA-sensing pattern recognition receptor" of the second component of the present combination is a double-stranded nucleic acid, where the nucleic acid comprises or consists of nucleotides selected from DNA nucleotides, RNA nucleotides, PNA nucleotides, and / or LNA nucleotides, or analogs of any of these.

[0086] As used herein, the term "LNA nucleotide" refers to a modified RNA nucleotide. LNA nucleotides are locked nucleic acids. The ribose moiety of an LNA nucleotide can be modified with an additional bridge connecting the 2'-oxygen and 4'-carbon. This bridge locks the ribose into the 3'-endo (North) conformation, which is often seen in A-form duplexes. LNA nucleotides can be mixed, for example, with DNA or RNA residues in an oligonucleotide. LNA nucleotides hybridize to DNA or RNA. Oligomers containing LNA nucleotides are chemically synthesized and commercially available. The locked ribose conformation enhances base stacking and pre-organization of the backbone.

[0087] As used herein, the term "PNA nucleotide" refers to a modified nucleic acid. DNA and RNA have a deoxyribose and ribose sugar backbone, respectively. The backbone of PNA is composed of a repeating unit of N-(2-aminoethyl)-glycine units, which are linked by peptide bonds. Thus, PNA is depicted like a peptide, i.e., from the N-terminus to the C-terminus. PNA exhibits higher binding strength. PNA oligomers also show greater specificity in binding to complementary DNA, and PNA / DNA base mismatches are less stable than similar mismatches in DNA / DNA duplexes. This binding strength and specificity also apply to PNA / RNA duplexes. PNA is not readily recognized by either nucleases or proteases, and PNA is also stable over a wide pH range.

[0088] In certain embodiments, the nucleic acid of the second component is a hybrid RNA nucleic acid, and the hybrid RNA nucleic acid comprises RNA nucleotides and further at least one DNA, LNA, or PNA nucleotide.

[0089] In certain embodiments, the nucleic acid comprises at least one modified nucleotide and / or at least one nucleotide analog or nucleotide derivative.

[0090] The terms "analog" or "derivative" can be used interchangeably to generally refer to any purine and / or pyrimidine nucleotide or nucleoside having a modified base and / or sugar. A modified base is a base other than guanine, cytosine, adenine, thymine, or uracil. A modified sugar is any sugar other than ribose or 2'-deoxyribose and can be used in the backbone of an oligonucleotide.

[0091] In an embodiment, the nucleic acid of the second component comprises at least one modified nucleotide and / or at least one nucleotide analog, and the at least one modified nucleotide and / or the at least one nucleotide analog is selected from a backbone-modified nucleotide, a sugar-modified nucleotide, and / or a base-modified nucleotide, or any combination thereof.

[0092] A backbone modification in the context of the present invention is a modification in which the phosphate of the nucleotide backbone is chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of the nucleotide. A base modification in the context of the present invention is a chemical modification of the base portion of the nucleotide.

[0093] In an embodiment, the nucleotide analogs / modifications that can be incorporated into the nucleic acid of the second component as described herein are preferably selected from the following: 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-thiocytidine-5'-triphosphate, 2'-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoazenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate,Xanthosine-5'-triphosphate. Particular reference is made to nucleotides for base modification selected from the group of base-modified nucleotides consisting of the following: 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine,and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine, 5’-O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, 5’-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoiso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine,8-Oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.,

[0094] In a preferred embodiment, at least one modified nucleotide and / or at least one nucleotide analog is selected from modified nucleotides found in bacterial tRNA. In a particularly preferred embodiment, at least one modified nucleotide and / or at least one nucleotide analog is selected from the following: 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-thionylcarbamoyladenosine, 2-methylthio-N6-thionylcarbamoyladenosine, N6-methyl-N6-thionylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2'-O-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysidine, 1-methylguanosine, 7-methylguanosine, 2'-O-methylguanosine, queuosine, epoxyqueuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, dihydrouridine, 5-methyluridine, 2'-O-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine´, 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine.

[0095] In a preferred embodiment, the nucleic acid of the second component comprises at least one 2'-substituted RNA nucleotide (ribonucleoside).

[0096] The term "2'-substituted ribonucleoside" generally includes ribonucleosides in which the hydroxyl group at the 2'-position of the pentose moiety is substituted to produce a 2'-substituted or 2'-O-substituted ribonucleoside. In certain embodiments, such a substitution is a lower hydrocarbyl group containing 1 to 6 saturated or unsaturated carbon atoms, a halogen atom, or an aryl group having 6 to 10 carbon atoms, where such a hydrocarbyl, or aryl group may be unsubstituted or may be substituted, for example, with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carboalkoxy, or amino groups.

[0097] In a preferred embodiment, the nucleic acid of the second component comprises at least one sugar-modified nucleotide. Preferably, the sugar-modified nucleotide is at least one 2'-ribose-modified (ribonucleoside) RNA nucleotide.

[0098] Examples of 2'-O-substituted ribonucleosides include, but are not limited to, 2'-amino, 2'-fluoro, 2'-allyl, 2'-O-alkyl and 2'-propargyl ribonucleosides, 2'-O-methyl ribonucleoside and 2'-O-methoxyethoxy ribonucleoside.

[0099] In particularly preferred embodiments, at least one 2'-ribose-modified RNA nucleotide of the nucleic acid of the second component is a 2'-O-methylated RNA nucleotide (2'-O-methyl ribonucleotide).

[0100] In a particularly preferred specific example, the nucleic acid of the second component contains at least one 2'-ribose-modified RNA nucleotide, and the at least one 2'-ribose-modified RNA nucleotide is a 2'-O-methylated RNA nucleotide. Preferably, the 2'-O-methylated RNA nucleotide is selected from 2'-O-methylated guanosine (Gm), 2'-O-methylated uracil (Um), 2'-O-methylated adenosine (Am), 2'-O-methylated cytosine (Cm), or a 2'-O-methylated analog of any of these nucleotides.

[0101] In a particularly preferred specific example, the nucleic acid of the second component contains at least one 2'-O-methylated RNA nucleotide, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-O-methylated RNA nucleotides, and the at least one or the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-O-methylated RNA nucleotides may be selected from 2'-O-methylated guanosine (Gm), 2'-O-methylated uracil (Um), 2'-O-methylated adenosine (Am), 2'-O-methylated cytosine (Cm), or a 2'-O-methylated analog of any of these nucleotides.

[0102] In a preferred embodiment, the nucleic acid of the second component contains at least one 2'-O-methylated RNA nucleotide, where preferably the at least one 2'-O-methylated RNA nucleotide is not located at the 5'-end and / or 3'-end of the nucleic acid.

[0103] In a preferred embodiment, the nucleic acid of the second component contains at least one or more trinucleotide M-X-Y motifs, where in the formula, M is selected from Gm, Um, or Am, preferably M is Gm, in the formula, X is selected from G, A or U, preferably X is G or A, in the formula, Y is selected from G, A, U, C or dihydrouridine, preferably Y is C.

[0104] In particularly preferred embodiments, the nucleic acid of the second component comprises at least one or more trinucleotide M-X-Y motifs, where wherein M is Gm, wherein X is G or A, wherein Y is C.

[0105] In certain embodiments, the nucleic acid of the second component comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more trinucleotide M-X-Y motifs as defined herein, where each M-X-Y motif may be independently defined as described herein.

[0106] In certain embodiments, the nucleic acid of the second component comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more trinucleotide M-X-Y motifs as defined herein, and the trinucleotide motifs are not located at the 3' and / or 5' ends.

[0107] In particularly preferred embodiments, the nucleic acid of the second component comprises or consists of at least one nucleic acid sequence according to formula I: N W -M-X-Y-N Z (Formula I) wherein N is independently selected from any nucleotide or nucleotide analog as defined herein, preferably G, A, U, C, Gm, Am, Um, Cm, or a modified nucleotide as defined herein; wherein W is 0 or an integer from 1 to 15, preferably W is an integer from 1 to 10, most preferably 1 to 5; wherein Z is 0 or an integer from 1 to 15, preferably Z is an integer from 1 to 10, most preferably 1 to 5; wherein M, X, and Y are selected as defined herein.

[0108] In particularly preferred embodiments, the nucleic acid of the second component comprises or consists of at least one nucleic acid sequence according to formula I: wherein N is independently selected from G, A, U, C; In the formula, W is an integer from 1 to 10; In the formula, Z is an integer from 1 to 10; In the formula, M is Gm; In the formula, X is G; In the formula, Y is C.

[0109] Exemplary nucleic acid sequences that can be derived from Formula I are as follows: 5’-MXYNNNNNNNN-3’ 5’-NMXYNNNNNNN-3’ 5’-NNMXYNNNNNN-3’ 5’-NNNMXYNNNNN-3’ 5’-NNNNMXYNNNN-3’ 5’-NNNNNMXYNNN-3’ 5’-NNNNNNMXYNN-3’ 5’-NNNNNNNMXYN-3’ 5’-NNNNNNNNMXY-3’ 5’-MXYNNNNNNN-3’ 5’-NMXYNNNNNN-3’ 5’-NNMXYNNNNN-3’ 5’-NNNMXYNNNN-3’ 5’-NNNNMXYNNN-3’ 5’-NNNNNMXYNN-3’ 5’-NNNNNNMXYN-3’ 5’-NNNNNNNMXY-3’ 5’-MXYNNNNNN-3’ 5’-NMXYNNNNN-3’ 5’-NNMXYNNNN-3’ 5’-NNNMXYNNN-3’ 5’-NNNNMXYNN-3’ 5’-NNNNNMXYN-3’ 5’-NNNNNNMXY-3’ 5’-MXYNNNNN-3’ 5’-NMXYNNNN-3’ 5’-NNMXYNNN-3’ 5’-NNNMXYNN-3’ 5’-NNNNMXYN-3’ 5’-NNNNNMXY-3’ etc.

[0110] In a particularly preferred specific example, the nucleic acid of the second component comprises, or consists of, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I, and each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I may be the same or may be selected independently of each other.

[0111] In that context, exemplary nucleic acid sequences that may be derived from formula I are as follows: 5’-NNNNNMXYMXYNNNNNNNNNNNMXYN-3’ 5’-NNNNNMXYMXYNNNNNNNNMXYN-3’ 5’-NNMXYNNNNNMXYNNNMXYNNN-3’ 5’-NNNMXYMXYNNNNNNNNNMXYN-3’ 5’-NNMXYNNNMXYNNNMXYNNN-3’ 5’-NNNMXYMXYNNNNNNMXYN-3’ 5’-MXYNNNNNNNNNNNNNMXY-3’ 5’-MXYNNNNNNNNNNNNNMXY-3’ 5’-NNMXYNNNNNMXYNNNMN-3’ 5’-MXYNNNNNNNNNNNMXY-3’ 5’-NNMXYNNNMXYNNNNN -3’ 5’-MXYNNNNNNNNNMXY-3’ etc.

[0112] In particularly preferred embodiments, the nucleic acid of the second component comprises a 5' end lacking a triphosphate group. In other words, the 5' end of the nucleic acid of the second component may comprise a monophosphate group or a diphosphate group or a hydroxyl group. The absence of a 5' triphosphate group in the nucleic acid of the second component is particularly important in the context of the present invention because such 5' ppp groups potentially stimulate the innate immune response (via RIG-1) upon administration.

[0113] Thus, in embodiments, the nucleic acid of the second component is generated using a synthetic method (e.g., RNA synthesis). In embodiments where the nucleic acid of the second component is generated using enzymatic treatment (e.g., RNA in vitro transcription), it may be necessary to remove the 5' ppp group of the nucleic acid in order to obtain a nucleic acid containing a 5' end lacking a triphosphate group (e.g., using phosphatase treatment).

[0114] In alternative embodiments, the nucleic acid of the second component contains a triphosphate group at the 5' end, where such 5' triphosphate group containing the nucleic acid may be generated using a synthetic method or enzymatic treatment.

[0115] The nucleic acid of the second component may have a length of from 1 to about 200 nucleotides, about 3 to about 200 nucleotides, about 3 to about 50 nucleotides, about 3 to about 25 nucleotides, about 5 to about 25 nucleotides, about 5 to about 15, or about 5 to about 10 nucleotides.

[0116] In preferred embodiments, the nucleic acid of the second component has a length of from about 3 to about 50 nucleotides, about 5 to about 25 nucleotides, about 5 to about 15, or about 5 to about 10 nucleotides. In particularly preferred embodiments, the nucleic acid of the second component has a length of from about 5 to about 15 nucleotides.

[0117] In various specific embodiments, the nucleic acid of the second component has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0118] In certain preferred embodiments, the nucleic acid of the second component has a length of 6, 7, 8, 9, 10, 11, or 12 nucleotides. Preferably, the nucleic acid of the second component has a length of 9 nucleotides.

[0119] In a preferred embodiment, the nucleic acid of the second component is a single-stranded oligonucleotide. In particularly preferred embodiments, the nucleic acid of the second component is a single-stranded RNA oligonucleotide.

[0120] RNA oligonucleotides in the context of the present invention include RNA nucleotides and preferably at least one chemically modified RNA nucleotide. RNA oligonucleotides are typically short RNA molecules having a length not exceeding 200 nucleotides. Typically, RNA oligonucleotides are chemically synthesized using building blocks of natural or chemically modified nucleosides and protected phosphoramidites.

[0121] The nucleoside residues of the oligonucleotide can be coupled to each other by any of a number of known internucleoside linkages. Such internucleoside linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carbalkoxy, acetamidoate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term "oligonucleotide" also includes polynucleosides having one or more stereospecific internucleoside linkages (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, or phosphotriester linkages). Preferred in the context of the present invention is the phosphodiester linkage.

[0122] An assembly of oligonucleotide chains proceeds in the 3' to 5' direction by following a routine procedure called a "synthesis cycle". When a single synthesis cycle is completed, one nucleotide residue is added to the growing chain. Thus, the second component nucleic acid is a single-stranded synthetic RNA oligonucleotide.

[0123] In some embodiments, the antagonist of the second component, preferably a nucleic acid, comprises two or more different nucleic acids, such as those defined herein as oligonucleotides, linked to a nucleotide or non-nucleotide linker called "branched" herein.

[0124] In some embodiments, the antagonist of the second component, preferably a nucleic acid, comprises two or more different nucleic acids for, e.g., an oligonucleotide as defined herein, and the two or more nucleic acids for the oligonucleotide are non-covalently linked, e.g., by electrostatic interactions, hydrophobic interactions, π-stacking interactions, hydrogen bonding, and combinations thereof. Non-limiting examples of such non-covalent bonds include Watson-Crick base pairing, Hoogsteen base pairing, and base stacking.

[0125] In some embodiments, the antagonist of the second component, preferably the nucleic acid antagonist, comprises a motif selected from CpG, C*pG*, and CpG*, where C is 2'-deoxycytidine, G is 2'-deoxyguanosine, C* is 2'-deoxythymidine, l-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, 5-Me-dC, 2'-di-deoxy-5-halocytosine, 2'-di-deoxy-5-nitrocytosine, arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, 2'-O-substituted ribonucleotide (including but not limited to 2'-O-Me-5-Me-C, 2'-O-(2-methoxyethyl)-ribonucleotide or 2'-O-Me-ribonucleotide) or other cytosine nucleotide derivatives, G* is 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabinoguanosine, 2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, 2'-O-substituted ribonucleotide (including but not limited to 2'-O-(2-methoxyethyl)-ribonucleotide; or 2'-O-Me-ribonucleotide) or other guanine nucleotide derivatives, and p is a internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate.

[0126] In some embodiments, the antagonist of the second component, preferably a nucleic acid, comprises 7-deazaguanosine (c7G) and at least one UpG-containing motif.

[0127] In the art, it has been shown that bacterial tRNATyr sequence fragments can function as TLR antagonists (Schmitt et al 2017 RNA 23:1344-135). Thus, in embodiments, the nucleic acid of the second component comprises or consists of a nucleic acid sequence derived from a bacterial tRNA sequence. Preferably, the nucleic acid sequence is or is derived from a bacterial tRNATyr sequence.

[0128] In embodiments, the nucleic acid of the second component comprises or consists of a nucleic acid sequence derived from a bacterial tRNATyr sequence, wherein the nucleic acid sequence is or is derived from the D-loop of tRNATyr. In preferred embodiments, the nucleic acid sequence is or is derived from the D-loop of Escherichia coli tRNATyr.

[0129] In preferred embodiments, the nucleic acid of the second component is an RNA oligonucleotide, which is a fragment of the D-loop of Escherichia coli tRNATyr, wherein the fragment has a length of about 5 to about 15 nucleotides, wherein the nucleic acid sequence comprises at least one 2'-O-methylated RNA nucleotide, preferably at least one M-X-Y motif, wherein optionally, the RNA oligonucleotide lacks a 5'-triphosphate end, and optionally, the M-X-Y motif is not located at the 3'-end of the RNA oligonucleotide.

[0130] In an embodiment of the present invention, the nucleic acid of the second component, preferably an oligonucleotide, is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85 to 165, or 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 a fragment of any of these sequences, or consists of the same. Further information regarding each of these suitable nucleic acid sequences may also be derived from the sequence listing, in particular, the content provided therein with the identification number <223>.

[0131] In a preferred embodiment of the present invention, the nucleic acid of the second component, preferably an oligonucleotide, 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 a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85 to 100, 149 to 165, or a fragment of any of these sequences, or consists of the same.

[0132] In a more preferred embodiment of the present invention, the nucleic acid of the second component, preferably an oligonucleotide, 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 a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85 to 87, 149 to 165, or a nucleic acid sequence provided in Table B, rows 1 to 20, or a fragment of any of these sequences, or consists of the same.

[0133] Particularly preferred in that context is 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 the nucleic acid sequence set forth in SEQ ID NO: 85, or provided in row 1 of Table B, or a fragment of any of these sequences.

[0134] In the following table (Table B), preferred nucleic acid sequences of the second component are provided, where modified nucleotides (e.g., Gm) are shown, and preferably, the sequences provided in Table B are RNA oligonucleotides. Particularly preferred is the RNA oligonucleotide 5'-GAG CGmG CCA-3' (see Table B, row 1), where the 5-position of the RNA oligonucleotide is 2'-O-methylated guanosine (Gm). Further information regarding each of these suitable nucleic acid sequences may also be derived from the sequence listing, in particular, the content provided therein with the identification number <223>.

[0135] [Table 2]

[0136] JPEG2025106391000003.jpg104169

[0137] In other embodiments of the present invention, the nucleic acid of the second component, preferably an oligonucleotide, is selected from the following: IRS-954 (DV-1079), IRO-5, IRS 2088, IRS 869, INH-ODN-2114, INH-ODN 4024, INH-ODN 4084-F, IRS-661, IRS-954, INH-ODN-24888, IHN-ODN 2088, ODN 20958, IHN-ODN-21595, IHN-ODN-20844, IHN-ODN-24991, IHN-ODN-105870, IHN-ODN-105871, ODN A151, G-ODN, ODN INH-1, ODN INH-18, ODN 4084-F, INH-4, INH-13, (pS-)ST-ODN, INH-ODN 21 14, CMZ 203-84, CMZ 203-85, CMZ 203-88, CMZ 203-88-1, CMZ 203-91, ODN 4084, ODN INH-47, CpG-52364 (quinazoline derivative from Coley Pharmaceutical), IMO-3100, IMO-8400, IMO-8503 (inhibitory RNA / DNA hybrid oligonucleotide), ODN 2087, ODN 20959, SM934, IMO-4200, IMO-9200, DV-1179, VTX-763, TMX-302, TMX-306, and further oligonucleotides disclosed in the following: Schmitt et al. (Schmitt et al 2017. RNA 23:1344-135.), Robbins et al. (Robbins et al 2007. Molecular therapy Vol 15 No 9, 1663-1669.), WO2008017473 (especially Tables 2 and 6, SEQ ID Nos 195-201), WO2009141146 (SEQ ID Nos 4-56), WO2010105819, US2009087388 (Tables 4 and 6), WO2017136399 (Table 4) and WO2008033432 (Tables 1-5 and 8).

[0138] In a further specific embodiment, the nucleic acid of the second component, preferably the oligonucleotide, is or is derived from the published PCT application WO2009055076, in particular claims 44 to 45 of WO2009055076. The disclosure of WO2009055076, in particular the disclosure regarding claims 44 to 45 of WO2009055076, is incorporated herein by reference.

[0139] 〔First component: Therapeutic RNA〕 Advantageous embodiments and features of at least one therapeutic RNA of the first component are described below. In particular, all embodiments and features of said therapeutic RNA described in the context of the combination of the present invention (first aspect) are equally applicable to the therapeutic RNA of the pharmaceutical composition (second aspect), or the partial kit (third aspect), and further aspects of the present invention.

[0140] In various embodiments, at least one therapeutic RNA of the first component is selected from coding RNA, non-coding RNA, circular RNA (circRNA), RNA oligonucleotide, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, mRNA, riboswitch, immunostimulatory RNA (isRNA), ribozyme, RNA aptamer, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA, small nuclear RNA (snRNA), self-replicating RNA, replicon RNA, small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0141] The term "RNA" is recognized and understood by those skilled in the art and is intended to be, for example, a ribonucleic acid molecule, i.e., a polymer consisting of nucleotides. These nucleotides are typically adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate, and cytidine-monophosphate monomers linked to each other along a so-called backbone. The backbone is typically formed by a phosphodiester bond between the sugar of the first monomer, i.e., ribose, and the phosphate moiety of the second adjacent monomer. The specific sequence of monomers is called an RNA sequence.

[0142] The term "therapeutic RNA" relates to any RNA that provides a therapeutic modality, particularly any RNA as defined above. The term "therapy" in this context must be understood as "providing a therapeutic function" or "being suitable for treatment or administration". However, "therapeutic" in that context should not at all be understood as being limited to a specific therapeutic modality. Examples of therapeutic modalities can be the provision of a coding sequence (via said therapeutic RNA) that encodes a peptide or a protein (wherein said peptide or protein has a specific therapeutic function, for example, an antigen for a vaccine or an enzyme for protein replacement therapy). A further therapeutic modality can be genetic engineering, where the RNA provides or modulates factors for manipulating DNA and / or RNA, for example. Typically, the term "therapeutic RNA" does not include natural RNA extracts or RNA preparations (such as those obtained from bacteria or plants) that are not suitable for administration to a subject (such as an animal, a human). For being suitable for therapeutic purposes, the RNA of the present invention may be an artificial, non-natural RNA.

[0143] Thus, in a preferred embodiment, at least one therapeutic RNA of the first component is an artificial RNA.

[0144] As used herein, the term "artificial RNA" is intended to refer to RNA that does not occur naturally. In other words, artificial RNA can be understood as a non-natural RNA molecule. Such RNA molecules can be non-natural due to their individual sequences (e.g., G / C content modified coding sequences, UTRs) and / or other modifications such as structural modifications of modified nucleotides. Artificial RNA can be designed and / or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, artificial RNA is a sequence that cannot occur naturally, i.e., a sequence that differs from the wild-type sequence by at least one nucleotide / modification.

[0145] In embodiments, at least one therapeutic RNA of the first component is preferably a non-coding RNA selected from RNA oligonucleotides, small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), antisense RNAs (asRNAs), CRISPR / Cas9 guide RNAs, riboswitches, ribozymes, RNA aptamers, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs).

[0146] In a preferred embodiment, at least one therapeutic RNA of the first component is a non-coding RNA, preferably a CRISPR / Cas9 guide RNA or a small interfering RNA (siRNA).

[0147] As used herein, the term "guide RNA" (gRNA) refers to any RNA molecule capable of targeting a CRISPR-associated protein / CRISPR-associated endonuclease to a target DNA sequence of interest. In the context of the present invention, the term guide RNA must be understood in its broadest sense and can include crRNA ("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA repeat") and the corresponding tracrRNA ("trans-acting CRISPR RNA" or "activator-RNA" or "tracrRNA") molecules or a two-molecule gRNA ("tracrRNA / crRNA") including a single-molecule gRNA. "sgRNA" typically includes a crRNA linked at its 3' end to the 5' end of the tracrRNA via a "loop" sequence. In the context of the present invention, the guide RNA may be provided by at least one therapeutic RNA of the combinations / compositions of the present invention.

[0148] In a preferred embodiment, at least one therapeutic RNA of the first component is a coding RNA. Most preferably, said coding RNA may be selected from mRNA, (coding) self-replicating RNA, (coding) circular RNA, (coding) viral RNA, or (coding) replicon RNA.

[0149] Coding RNA can be any type of RNA construct (e.g., double-stranded RNA, single-stranded RNA, circular double-stranded RNA, or circular single-stranded RNA) characterized in that the coding RNA contains at least one sequence (cds) that is translated into at least one amino acid sequence (e.g., upon administration to a cell).

[0150] As used herein, the terms "coding sequence", "coding region", or "cds" are intended to refer, for example, to a sequence of several nucleotides that can be recognized and understood by one of ordinary skill in the art and translated into a peptide or protein. In the context of the present invention, a cds is preferably an RNA sequence consisting of a number of nucleotide triplets that preferably begin with a start codon and preferably end with one stop codon. In embodiments, the cds of RNA can end with one or two or more stop codons. The first stop codon of two or more stop codons may be TGA or UGA, and the second stop codon of two or more stop codons may be selected from TAA, TGA, TAG, UAA, UGA or UAG.

[0151] In embodiments, at least one therapeutic RNA of the first component is a circular RNA. As used herein, "circular RNA" or "circRNA" should be understood as a circular polynucleotide construct that can encode at least one peptide or protein. Thus, in preferred embodiments, the circRNA comprises at least one cds encoding at least one peptide or protein as defined herein. CircRNAs can be synthesized using a variety of methods available in the art, including, for example, the methods provided in U.S. Patent No. 6210931, U.S. Patent No. 5773244, International Publication No. 1992 / 001813, International Publication No. 2015 / 034925, and International Publication No. 2016 / 011222, which are incorporated herein by reference.

[0152] In an embodiment, at least one therapeutic RNA of the first component is a replicon RNA. The term "replicon RNA" is recognized and understood by those skilled in the art and is intended to be, for example, an optimized self-replicating RNA. Such constructs can include, for example, replicase elements derived from an alphavirus (such as SFV, SIN, VEE, or RRV), as well as substitutions of the nucleic acid of the structural viral protein with the nucleic acid of interest, and coding sequences. Alternatively, the replicase can be provided on an independent RNA construct. Downstream of the replicase can be a subgenomic promoter that controls the replication of the replicon RNA.

[0153] In a particularly preferred embodiment, at least one therapeutic RNA of the first component is a messenger RNA (mRNA). A typical mRNA (messenger RNA) in the context of the present invention provides a coding sequence that is translated, for example, into the amino acid sequence of a peptide or protein after in vivo administration to a cell.

[0154] In a preferred embodiment, at least one therapeutic RNA of the first component, particularly the coding RNA or mRNA, is an in vitro transcribed RNA. In that context, appropriately, the therapeutic RNA is an in vitro transcribed coding RNA or an in vitro transcribed mRNA.

[0155] RNA in vitro transcribed RNA should be understood as RNA obtained by RNA in in vitro transcription.

[0156] The term "RNA in vitro transcription" or "in vitro transcription" relates to the process by which RNA is synthesized in a cell-free system (in vitro). RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which is a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerases. In a preferred embodiment, the DNA template is linearized with an appropriate restriction enzyme before it is subjected to RNA in vitro transcription.

[0157] Reagents typically used for RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) having a promoter sequence with high binding affinity for each RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5);, where appropriate, a cap analog as defined herein; where appropriate, a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) that can bind to the promoter sequence within the DNA template; where appropriate, a ribonuclease (RNase) inhibitor that inactivates potentially contaminating RNases; where appropriate, a pyrophosphatase that degrades pyrophosphate; a buffer (Tris or HEPES) that supplies Mg2+ ions as a cofactor for the polymerase, an antioxidant (e.g., DTT), and / or a polyamine such as spermidine, at optimal concentrations, in a system containing, for example, a buffer, such as Tris-citrate, as disclosed in WO2017 / 109161.

[0158] Thus, in a preferred embodiment, at least one therapeutic RNA of the first component, particularly the coding RNA or mRNA, is RNA in vitro transcription RNA, where the RNA in vitro transcription RNA can be obtained by RNA in vitro transcription using a sequence-optimized nucleotide mixture.

[0159] In that context, the nucleotide mixture used in in vitro RNA transcription may further contain modified nucleotides as defined below. In a preferred embodiment, the nucleotide mixture used in the in vitro RNA transcription reaction (i.e., the fraction of each nucleotide in the mixture) is preferably optimized essentially for a given RNA sequence (optimized NTP mixture) as described in WO2015 / 188933. The RNA obtained by treatment with the optimized NTP mixture is characterized by a reduced immunostimulatory property.

[0160] In a preferred embodiment, at least one therapeutic RNA of the first component, particularly the coding RNA or mRNA, is purified RNA (e.g., purified in vitro transcribed mRNA).

[0161] As used herein, the term "purified RNA" must be understood as a therapeutic RNA having a higher purity than the starting material (e.g., in vitro transcribed RNA or synthetic RNA) after specific purification steps (e.g., (RP)-HPLC, TFF, oligo d(T) purification, precipitation steps). Typical impurities that are not essentially presented in the purified RNA include peptides or proteins (e.g., for enzymes derived from in vitro transcription, RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, incomplete RNA sequences, RNA fragments (short double-stranded RNA fragments, incomplete sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that may be derived from fermentation procedures include bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents, etc.). Thus, in this regard, it is desirable that the "degree of RNA purity" be as close to 100% as possible. It is also desirable for the amount of full-length RNA transcript to be as close to 100% as possible in terms of the degree of RNA purity. Thus, "purified RNA" as used herein has a purity of 70%, 80%, 85%, very particularly 90%, 95%, most preferably 99% or more. Further, "purified RNA" as used herein may further or alternatively have an amount of full-length RNA of 70%, 80%, 85%, very particularly 90%, 95%, and most preferably 99% or more. Such purified RNA as defined herein is characterized by a reduced immunostimulatory property (compared to non-purified RNA), which is particularly preferred in the context of this specification.

[0162] The purity or amount of full-length RNA can be determined, for example, by analytical HPLC, where the percentages provided above correspond to the ratio between the area of the peak for the desired RNA and the total area of all peaks in the chromatogram. Alternatively, the degree of purity can be determined by other methods, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0163] Particularly for medical applications, it may be necessary to provide pharmaceutical-grade RNA. In particularly preferred embodiments, RNA production is carried out under current Good Manufacturing Practice (GMP), and various quality control steps for DNA and RNA concentrations are preferably implemented according to the procedures as described in WO2016 / 180430. The resulting RNA product is preferably purified using RP-HPLC (as described in WO2008 / 077592) and / or tangential flow filtration (as described in WO2016 / 193206). Thus, in a preferred embodiment, at least one therapeutic RNA of the first component, particularly coding RNA or mRNA, is GMP-grade RNA or pharmaceutical-grade RNA.

[0164] In a preferred embodiment, at least one therapeutic RNA of the first component, particularly coding RNA or mRNA, is purified RNA (for example, purified in vitro transcribed mRNA), and the purified RNA is purified by RP-HPLC and / or TFF and / or Oligo d(T) purification. Preferably, the purified RNA is (RP)-HPLC purified RNA.

[0165] "Purified RNA" as defined herein or "pharmaceutical-grade RNA" as defined herein may have excellent stability properties (in vitro, in vivo) and improved efficacy (e.g., better translatability of RNA in vivo), and thus it must be emphasized that it is particularly suitable for any medical application. Furthermore, such RNA is characterized by reduced immunostimulatory properties (compared to non-purified RNA), which is preferred in the context of this specification.

[0166] In certain embodiments, at least one therapeutic RNA of the first component, particularly coding RNA or mRNA, is in vitro transcribed RNA, purified RNA, pharmaceutical-grade RNA. Such RNA is characterized by reduced immunostimulatory properties (compared to, for example, non-purified in vitro transcribed RNA), and thus is particularly suitable in the context of the present invention.

[0167] In a preferred embodiment, at least one therapeutic RNA of the first component, such as a coding RNA or mRNA, comprises at least one coding sequence (cds) encoding at least one peptide or protein.

[0168] Advantageously, the expression of at least one peptide or protein encoded by the coding RNA or mRNA is increased or extended by combination with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component upon administration to a cell, tissue or organism, compared to the expression of at least one peptide or protein encoded by the coding RNA or mRNA without combination with at least one antagonist of at least one RNA-sensing receptor of the second component.

[0169] Thus, the combined administration to a cell, tissue, or organism (i.e., the administration of the first and second components) results in an increase or extension of peptide / protein expression compared to the administration of the corresponding first component / therapeutic RNA alone.

[0170] Methods for assessing the expression (i.e., protein expression) of therapeutic RNA in a specific cell / organ / tissue and for determining the duration of expression are well known to those skilled in the art. For example, protein expression can be determined using antibody-based detection methods (western blot, FACS) or quantitative mass spectrometry. Exemplary methods are provided in the Examples section. Typically, the expression of the therapeutic RNA in combination with the second component is compared to the expression of the therapeutic RNA alone (or the first component alone), i.e., without the (additional) administration of the second component. The same conditions (e.g., the same cell line, the same organism, the same application route, the same detection method, the same amount of therapeutic RNA, the same RNA sequence) should be used (if possible) to enable a valid comparison. Those skilled in the art understand how to perform the comparison of the combinations of the present invention and the respective control RNAs (e.g., therapeutic RNA only or first component only).

[0171] The "increased protein expression" of the combinations of the present invention should be understood as the percentage increase in expression as determined by various well-established expression assays (such as antibody-based detection methods) as described above, compared to the corresponding control (either the first component only or the therapeutic RNA only).

[0172] Thus, administration of the combination to a cell, tissue, or organism (i.e., administration of the first and second components) results in an increase in expression compared to administration of the corresponding first component / therapeutic RNA only, where the percent increase in expression in said cell, tissue, or organism is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more.

[0173] The "extended protein expression" of the combinations of the present invention should be understood as the further duration of protein expression such that the expression of the combinations of the present invention is still detectable, compared to the corresponding control (either the first component only or the therapeutic RNA only) as determined by various well-established expression assays (such as antibody-based detection methods) as described above.

[0174] Thus, administration of the combination to a cell, tissue, or organism (i.e., administration of the first and second components) results in extended protein expression compared to administration of the corresponding first component / therapeutic RNA only, where the further duration of protein expression 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 10h or more.

[0175] In particularly preferred embodiments, the expression of at least one peptide or protein encoded by the coding RNA or mRNA is increased or extended upon administration to a cell, tissue or organism, in combination with at least one antagonist of at least one RNA-sensing receptor of the second component, as compared to the expression of at least one peptide or mRNA encoded. At the same time, however, without combining with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component, the administration of at least one coding RNA or the combination of at least one RNA-sensing pattern recognition receptor of the second component and at least one antagonist of at least one RNA-sensing pattern recognition receptor results in a decrease in the innate immune response as compared to the administration of at least one coding RNA or mRNA of the first component without combining with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component.

[0176] In a preferred embodiment, the cds of the coding RNA or mRNA encodes at least one peptide or protein, where the at least one peptide or protein is or is derived from a therapeutic peptide or protein.

[0177] In various embodiments, the length of the peptide or protein encoded, such as a therapeutic peptide or protein, may be at least or about 20, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1500 amino acids.

[0178] In embodiments, at least one therapeutic peptide or protein is an antibody, in vivo, receptor, agonist, receptor antagonist, binding protein, CRISPR-associated endonuclease, chaperone, transporter protein, ion channel, membrane protein, secreted protein, transcription factor, enzyme, peptide or protein, growth factor, structural protein, cytoplasmic protein, cytoskeletal protein, viral antigen, bacterial antigen, protozoal antigen, allergen, tumor antigen, or a fragment or variant of any of these, or is derived therefrom.

[0179] In some embodiments, the antibodies encoded by the RNA or mRNA of the present invention can be selected from all antibodies, for example, produced by recombinant methods or naturally occurring and known to those skilled in the art from the prior art, all antibodies, in particular, antibodies used (usable) for therapeutic, diagnostic or investigational purposes, or antibodies found for specific diseases such as cancer diseases, infectious diseases, etc., as also described in WO2008083949 which is incorporated herein by reference and can be selected from.

[0180] In the context of the present invention, the antibodies encoded by the RNA or mRNA according to the present invention typically include all antibodies known to those skilled in the art, for example, naturally occurring antibodies, or antibodies produced in a host organism by immunization, antibodies prepared by recombinant methods isolated and identified from naturally occurring antibodies, or antibodies produced in a host organism by (conventional) immunization, or antibodies produced with the aid of molecular biological methods, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e., antibodies expressed in cells and perhaps localized in specific cell compartments, and fragments of the above-mentioned antibodies. To that extent, the term antibody should be understood in its broadest sense. In this context, an antibody generally typically includes a light chain and a heavy chain, both of which have variable domains and constant domains.

[0181] According to an embodiment, the cds of at least one therapeutic RNA as defined herein encodes at least one (therapeutic) peptide or protein as defined above, and further at least one additional heterologous peptide or protein element.

[0182] Suitably, the at least one additional heterologous peptide or protein element may be selected from a secretion signal peptide, a transmembrane element, a multimerization domain, a VLP-forming sequence, a nuclear localization signal (NLS), a peptide linker element, a self-cleaving peptide, an immunological adjuvant sequence or a dendritic cell targeting sequence.

[0183] According to a preferred embodiment, the therapeutic RNA of the first component comprises at least one cds, and the cds encodes at least one peptide or protein as specified herein. In this context, any cds encoding at least one peptide or protein may be understood as a suitable cds and thus may be included in the therapeutic RNA.

[0184] In an embodiment, the length of the cds may be longer than at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, 5000, or 6000 nucleotides. In an embodiment, the length of the cds may range from about 300 to about 2000 nucleotides.

[0185] In a preferred embodiment, the therapeutic RNA of the first component is a modified and / or stabilized RNA, preferably a modified and / or stabilized coding RNA or a modified and / or stabilized mRNA.

[0186] Thus, the therapeutic RNA of the first component can be provided as "stabilized artificial RNA", i.e., RNA that exhibits improved resistance to in vivo degradation and / or RNA that exhibits improved stability in vivo and / or RNA that exhibits improved translatability in vivo.

[0187] The following describes modifications suitable for "stabilizing" the therapeutic RNA of the first component.

[0188] In a preferred embodiment, at least one cds of the therapeutic RNA of the first component is a codon-modified cds, where the amino acid sequence encoded by at least one codon-modified cds is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type cds.

[0189] The term "codon-modified coding sequence" relates to a coding sequence in which at least one codon (a triplet of nucleotides encoding one amino acid) is different compared to the corresponding wild-type cds. The codon-modified cds in the context of the present invention exhibits improved resistance to in vivo degradation and / or improved stability in vivo and / or improved translatability in vivo. Since multiple codons encoding the same amino acid can be used interchangeably to optimize / modify the coding sequence, codon modification utilizes the degeneracy of the genetic code (Table 1).

[0190] In particularly preferred embodiments, at least one cds of the therapeutic RNA of the first component is a codon-modified cds, where the codon-modified cds is selected from C-maximized cds, CAI-maximized cds, human codon usage frequency-conforming cds, G / C content-modified cds, and G / C-optimized cds, or any combination thereof.

[0191] In a preferred embodiment, the therapeutic RNA of the first component can be modified to increase the C content of at least one cds as compared to the C content of the corresponding wild-type cds (referred to herein as the "C-maximized coding sequence"), preferably to maximize it. The amino acid sequence encoded by the C-maximized cds is preferably not modified as compared to the amino acid sequence encoded by each wild-type nucleic acid cds. The generation of the C-maximized nucleic acid sequence can be carried out using the method according to WO2015 / 062738 (the disclosure of WO2015 / 062738 is incorporated herein by reference).

[0192] In an embodiment, the therapeutic RNA of the first component can be modified to modify the G / C content of at least one cds as compared to the G / C content of the corresponding wild-type cds (referred to herein as the "G / C content-modified coding sequence"). In this context, the terms "G / C optimization" or "G / C content modification" relate to an RNA containing a modified, preferably increased number of guanosine and / or cytosine nucleotides as compared to the corresponding wild-type RNA. Such an increased number can be generated by substituting codons containing A or T nucleotides with codons containing G or C nucleotides. Advantageously, an RNA sequence having an increased G / C content is more stable than the corresponding wild-type sequence or a sequence having an increased A / U content (which can result in increased translation in vivo). The amino acid sequence encoded by the G / C content-modified cds is preferably not modified as compared to the amino acid sequence encoded by each wild-type sequence. Preferably, the G / C content of at least one cds is increased by at least 10%, 20%, 30%, preferably at least 40% as compared to the G / C content of the cds of the corresponding wild-type sequence.

[0193] In a preferred embodiment, the therapeutic RNA of the first component can be modified, and the G / C content of at least one cds can be optimized as compared to the G / C content of the corresponding wild-type cds (referred to herein as "G / C content optimized coding sequence"). "Optimized" in this context refers to a cds in which the G / C content is preferably increased to essentially the highest G / C content. The amino acid sequence encoded by the G / C content optimized cds is preferably not modified as compared to the amino acid sequence encoded by each wild-type cds. Advantageously, an RNA sequence having a G / C content optimized coding sequence is more stable than the corresponding wild-type sequence (which can lead to increased translation in vivo). Generation of the G / C content optimized coding sequence can be carried out according to WO2002 / 098443 (the disclosure of WO2002 / 098443 is incorporated herein by reference).

[0194] In an embodiment, the therapeutic RNA of the first component can be modified, and the codons in at least one cds can be adapted to human codon usage frequency (referred to herein as "human codon usage frequency adapted coding sequence"). Codons encoding the same amino acid are present in various frequencies in a subject (e.g., human). Thus, the cds is preferably modified such that the frequency of codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to human codon usage frequency. For example, for the amino acid Ala, it is preferred to adapt the wild-type cds to use the codon "GCC" at a frequency of 0.40, the codon "GCT" at a frequency of 0.28, the codon "GCA" at a frequency of 0.22, the codon "GCG" at a frequency of 0.10, etc. (see Table 1). Thus, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the cds to obtain a sequence adapted to human codon usage frequency. Advantageously, an RNA sequence having a human codon usage frequency adapted coding sequence can be more stable in vivo or exhibit better translational ability than the corresponding wild-type sequence.

[0195]

Table 3

[0196] In an embodiment, the therapeutic RNA of the first component can be modified such that the codon adaptation index (CAI) can be increased or preferably maximized in at least one cds (referred to herein as the "CAI-maximized coding sequence"). Thus, for example, it is preferred that all codons of the wild-type nucleic acid sequence that are relatively rare in human cells are replaced with the respective codons that are frequent in human cells, where the frequent codons encode the same amino acid as the relatively rare codons. Suitably, the most frequent codons are used for each encoded amino acid (see Table 1, the most frequent human codons are marked with a star). Suitably, the RNA comprises at least one cds and the codon adaptation index (CAI) of at least one cds is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of at least one cds is 1. For example, in the case of the amino acid Ala, the wild-type cds is adapted such that the most frequent human codon "GCC" is always used for said amino acid. Thus, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the cds to obtain the CAI-maximized cds.

[0197] In an embodiment, the therapeutic RNA (coding RNA or mRNA) of the first component can be modified by the addition of a 5'-cap structure that preferably stabilizes the RNA and / or enhances the expression of the encoded peptide or protein. The 5'-cap structure is particularly important in embodiments where the therapeutic RNA is linear, such as linear mRNA or linear replicon RNA. Thus, in a preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, comprises a 5'-cap structure.

[0198] In a preferred embodiment, the 5'-cap structure is an m7G (m7G(5')ppp(5')G), cap0, cap1, cap2, modified cap0 or modified cap1 structure.

[0199] As used herein, the term "5'-cap structure" is recognized and understood by those skilled in the art and is intended to refer to, for example, a 5'-modified nucleotide located at the 5'-end of an RNA, such as an mRNA, particularly a guanine nucleotide. Typically, the 5'-cap structure is linked to the RNA via a 5'-5'-triphosphate bond.

[0200] Suitable 5'-cap structures in the context of the present invention are cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0201] The 5'-cap (cap0 or cap1) structure can be formed in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using a cap analog.

[0202] As used herein, the term "cap analog" is recognized and understood by those skilled in the art and is intended to refer to non-polymeric dinucleotides or trinucleotides that have cap functionality in that, for example, when incorporated at the 5' end of a nucleic acid molecule, they facilitate translation or localization and / or prevent degradation of the nucleic acid molecule, particularly an RNA molecule. By non-polymeric is meant that they do not have a 5' triphosphate and cannot be extended in the 3' direction by template-dependent RNA polymerase, meaning that the cap analog is incorporated only at the 5' end. Examples of cap analogs include m7GpppG, m7GpppA, m7GpppC; non-methylated cap analogs (e.g., in the case of GpppG); dimethylated cap analogs (e.g., in the case of m2,7GpppG), trimethylated cap analogs (e.g., in the case of m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., in the case of m7Gpppm7G), or anti-reverse cap analogs (e.g., in the case of ARCA; m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, m7,3’dGpppG and their tetraphosphate derivatives), but are not limited thereto. Further cap analogs have been previously described (WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475). Further suitable cap analogs in that context are described in WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 053297, WO2017 / 066782, WO2018 / 075827 and WO2017 / 066797, the disclosures of which are incorporated herein by reference with respect to the cap analogs. Preferred cap analogs are the dinucleotide cap analog m7G(5’)ppp(5’)G (m7G) or 3’-O-Me-m7G(5’)ppp(5’)G, which co-transcriptionally generates a cap0 structure.

[0203] In an embodiment, the modified cap1 structure is generated using trinucleotide cap analogs as disclosed in WO2017 / 053297, WO2017 / 066793, WO2017 / 066781, WO2017 / 066791, WO2017 / 066789, WO2017 / 066782, WO2018 / 075827, and WO2017 / 066797. In particular, any cap structure derived from the structures disclosed in claims 1-5 of WO2017 / 053297 can be appropriately used to co-transcriptionally generate the modified cap1 structure. Further, any cap structure derivable from the structures defined in claim 1 or claim 21 of WO2018075827 can be appropriately used to co-transcriptionally generate the modified cap1 structure.

[0204] In a particularly preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, comprises a cap1 structure. (For example, using m7G(5’)ppp(5’)(2’OMeA)pG, or m7G(5’)ppp(5’)(2’OMeG)pG analogs,) the cap1 construct can be formed enzymatically or co-transcriptionally. The cap1 structure comprising RNA, preferably mRNA, includes an increase in the translation stimulation device and a decrease in the innate immune system and has several beneficial features.

[0205] In a preferred embodiment, the 5’-cap structure can be appropriately added co-transcriptionally using trinucleotide cap analogs as defined herein in an RNA in vitro transcription reaction as defined herein. It is advantageous that the RNA of the first component comprises a cap1 structure, and that the cap1 structure can be obtained by co-transcriptional capping.

[0206] In a preferred embodiment, the cap1 structure of at least one therapeutic RNA is formed using co-transcriptional capping using the trinucleotide cap analog m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG. The preferred cap1 analog in that context is m7G(5’)ppp(5’)(2’OMeA)pG.

[0207] Without being bound by theory, the beneficial effects of generating a capping 1 structure using co-transcriptional capping can be explained by improved capping efficiency compared to enzymatic capping, and / or by the fact that enzymatic capping can also generate intermediate capping 1 structures (e.g., partial methylation of the 5’ cap and / or a portion of the ribose following the 5’ cap).

[0208] In other embodiments, the 5’-cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2’-O-methyltransferase) to generate a capping 0 or capping 1 or capping 2 structure. The 5’-cap structure (cap0 or cap1) can be added using the methods and means disclosed in WO2016 / 193226 using an immobilized capping enzyme and / or cap-dependent 2’-O-methyltransferase.

[0209] In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the therapeutic RNA(s) of the first component comprises a cap 1 structure as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the therapeutic RNA(s) of the first component does not comprise a cap 1 structure as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the therapeutic RNA(s) of the first component comprises a cap 0 structure as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the coding RNA(s) of the first component comprises a cap 1 intermediate structure as determined using a capping assay.

[0210] The term "therapeutic RNA species" is not limited to meaning "one single molecule" and is understood to include a collection of essentially identical RNA therapeutic molecules. This term can preferably relate to multiple essentially identical coding RNA molecules that encode the same amino acid sequence.

[0211] To determine the capping level or the presence of a cap 1 intermediate, a capping assay as described in the published PCT application WO2015101416, in particular as described in claims 27-46 of the published PCT application WO2015101416, can be used. Other capping assays that can be used to determine the capping level of therapeutic RNA are described in PCT / EP2018 / 08667, or in the published PCT applications WO2014 / 152673 and WO2014152659.

[0212] In a preferred embodiment, the therapeutic RNA (coding RNA or mRNA) of the first component comprises a 5'-terminal m7G(5')ppp(5')(2'OMeA) cap structure. In such an embodiment, the RNA comprises a 5'-terminal m7G cap and further methylation of the ribose of the adjacent nucleotide of m7GpppN (in this case, 2'-O-methylated adenosine).

[0213] In other preferred embodiments, the therapeutic RNA (coding RNA or mRNA) of the first component comprises an m7G(5')ppp(5')(2'OMeG) cap structure. In such an embodiment, the RNA comprises a 5'-terminal m7G cap and further methylation of the ribose of the adjacent nucleotide (in this case, 2'-O-methylated guanosine).

[0214] Thus, whenever reference is made to a therapeutic coding RNA in the context of the present invention, the first nucleotide of said coding RNA or mRNA sequence, i.e., the nucleotide downstream of the m7G(5')ppp structure, can be 2'-O-methylated guanosine or 2'-O-methylated adenosine.

[0215] The stability or effectiveness of the RNA can also be provided, for example, by a modified phosphate backbone of the therapeutic RNA of the first component. The backbone modification may be a modification in which the phosphate of the backbone of the nucleotide of the RNA is chemically modified. Nucleotides that can be preferably used include, for example, phosphorothioate-modified phosphate backbones, preferably including that at least one of the phosphoric acid oxygens contained in the phosphate backbone is substituted by a sulfur atom. Stabilized RNAs can further include, for example, non-ionic phosphoric acid analogs such as alkyl and aryl phosphonates in which the charged phosphonate oxygen is replaced by an alkyl group or an aryl group, or phosphodiesters and alkyl phosphotriesters in which the charged oxygen residue is present in an alkylated form. Such backbone modifications typically include modifications from the group consisting of methylphosphonate, phosphoramidate, and phosphorothioate (e.g., cytidine-5'-O-(1-thiophosphate)).

[0216] Thus, in a preferred embodiment, at least one therapeutic RNA of the first component comprises at least one modified nucleotide and / or at least one nucleotide analog.

[0217] In an embodiment, at least one therapeutic RNA of the first component comprises at least one modified nucleotide, and the at least one modified nucleotide is selected from backbone-modified nucleotides, sugar-modified nucleotides and / or base-modified nucleotides or any combination thereof.

[0218] A backbone modification in the context of the present invention is a modification in which the phosphate of the nucleotide backbone is chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of the nucleotide of the RNA. A base modification in the context of the present invention is a chemical modification of the base portion of the nucleotide of the RNA. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs / modified nucleotides applicable to transcription and / or translation. Preferably, a nucleotide analog / modified nucleotide is selected that exhibits a reduced stimulation device of the innate immune system (after in vivo administration of an RNA comprising such a modified nucleotide).

[0219] In embodiments, the nucleotide analogs / modifications that can be incorporated into RNA as described herein are preferably selected from the following: 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propyl-2'-deoxycytidine-5'-triphosphate, 5-propyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate,Xanthosine-5'-triphosphate. Particular reference is made to nucleotides for base modification selected from the group of base-modified nucleotides consisting of the following: 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propyl-uridine, 1-propyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine,and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine, 5’-O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, 5’-O-(1-thiophosphate)-pseudouridine, 6-azacytidine, 2-thio-cytidine, α-thio-cytidine, pseudoiso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-azauridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0220] In embodiments, at least one chemical modification is selected from: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0221] In embodiments, 100% of the uracils in the cds of the therapeutic RNA of the first component have a chemical modification, preferably a chemical modification at the 5-position of uracil. In other embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the uracil nucleotides in the cds have a chemical modification, preferably a chemical modification at the 5-position of said uracil nucleotide. Such modifications are suitable in the context of the present invention because the reduction of native uracil can potentially cause a reduction in the stimulation of the innate immune system (upon administration of the RNA containing such modified nucleotides to cells), which can occur after in vivo administration of the RNA.

[0222] Suitably, the therapeutic RNA of the first component, in particular the cds of said therapeutic RNA, can contain at least one modified nucleotide, where said at least one modified nucleotide can be selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine, where pseudouridine (ψ) is preferred.

[0223] In the context of the present invention, the therapeutic RNA of the first component, preferably mRNA, contains a 5'-cap structure as defined herein, preferably a Cap1 structure, and preferably contains no modified nucleotides as defined herein. Thus, the therapeutic RNA of the first component can contain a 5'-cap structure and an RNA sequence containing A, U, G, C nucleotides, where the RNA sequence lacks any modified nucleotides.

[0224] In an alternative embodiment, the therapeutic RNA of the first component, preferably mRNA, contains a 5'-cap structure as defined herein, preferably a Cap1 structure, and further preferably contains a modified nucleotide as defined herein selected from pseudouridine (ψ), N1-methylpseudouridine (M1ψ), 5-methylcytosine, and 5-methoxyuridine.

[0225] In an embodiment, the A / U content in the sequence environment of the ribosome binding site of the therapeutic (coding) RNA can be increased compared to the A / U content in the environment of the ribosome binding site of its respective wild-type nucleic acid. This modification (increase in A / U content around the ribosome binding site) enhances the ability of the ribosome to bind to the RNA. The effective binding of the ribosome to the ribosome binding site then has the effect of efficient translation of the RNA.

[0226] Thus, in particularly preferred specific examples, the therapeutic (coding) RNA of the first component includes a ribosome binding site, also called a "Kozak sequence", that is identical or at least 80%, 85%, 90%, 95% identical to any one of the sequences of SEQ ID NO: 3 or 4, or a fragment or variant thereof.

[0227] In a preferred embodiment, at least one therapeutic RNA of the first component, preferably mRNA, includes at least one poly(A) sequence, and / or at least one poly(C) sequence, and / or at least one histone stem-loop sequence / structure.

[0228] Thus, the therapeutic (coding) RNA of the first component may include at least one poly(N) sequence, for example, at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or a combination thereof.

[0229] In a preferred embodiment, the therapeutic (coding) RNA includes at least one poly(A) sequence.

[0230] As used herein, the terms "poly(A) sequence", "poly(A) tail" or "3'-poly(A) tail" are recognized and understood by those skilled in the art and are intended to be, for example, a sequence of adenosine nucleotides up to about 1000, typically located at the 3' end of the coding RNA. The poly(A) sequence is essentially a homopolymer. For example, a poly(A) sequence for 100 adenosine nucleotides, for example, essentially has a length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from adenosine nucleotides.

[0231] The poly(A) sequence is appropriately positioned downstream of the 3’ UTR as defined herein and may contain from about 10 to about 500 adenosine nucleotides, from about 30 to about 500 adenosine nucleotides, from about 30 to about 200 adenosine nucleotides, or from about 50 to about 150 adenosine nucleotides. Suitably, the length of the poly(A) sequence may be at least about 30, 50, 64, 75, 100, 200, 300, 400, or more than 500 adenosine nucleotides. In a preferred embodiment, the poly(A) sequence contains from about 50 to about 250 adenosines. In particularly preferred embodiments, the poly(A) sequence contains about 64 adenosine nucleotides. In particularly preferred embodiments, the poly(A) sequence contains about 100 adenosine nucleotides.

[0232] The poly(A) sequence as defined herein is appropriately positioned at the 3’ end of the therapeutic RNA (e.g., mRNA). Thus, the 3’ terminal nucleotide of the RNA (i.e., the last 3’ terminal nucleotide of the polynucleotide chain) is preferably the 3’ terminal A nucleotide of at least one poly(A) sequence. The term “positioned at the 3’ end” must be understood to be precisely at the 3’ end, in other words, the 3’ end of the RNA consists of a poly(A) sequence that terminates in an A nucleotide.

[0233] Preferably, the poly(A) sequence of the therapeutic RNA of the first component is obtained from a DNA template during in vitro transcription of the RNA. In other embodiments, the poly(A) sequence is obtained in vitro by general methods of chemical synthesis without necessarily being transcribed from a DNA template. In other embodiments, the poly(A) sequence is generated by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocols known in the art, or alternatively, by using immobilized poly(A) polymerase using methods and means such as those described in WO 2016 / 174271.

[0234] Thus, the therapeutic RNA can contain a poly(A) sequence obtained by enzymatic polyadenylation, where most of the RNA molecule contains from about 100 (+ / -10) to about 500 (+ / -50), preferably about 250 (+ / -25) adenosine nucleotides.

[0235] In embodiments, the therapeutic RNA can contain a poly(A) sequence derived from a template DNA and can also contain at least one additional poly(A) sequence generated by enzymatic polyadenylation, as described in WO2016 / 091391.

[0236] In embodiments, the therapeutic RNA of the first component can contain at least one poly(C) sequence.

[0237] In embodiments, the poly(C) sequence is suitably located at or near the 3' end and contains from about 10 to 200 cytosine nucleotides, from about 10 to 100 cytosine nucleotides, or from about 10 to 50 cytosine nucleotides. In preferred embodiments, the poly(C) sequence contains about 30 cytosine nucleotides.

[0238] In preferred embodiments, the therapeutic RNA of the first component contains at least one histone stem-loop.

[0239] As used herein, the term "histone stem-loop" (abbreviated as "hsl") is intended to refer to a nucleic acid sequence that is recognized and understood by those skilled in the art and is found mainly in histone mRNA, for example.

[0240] The histone stem-loop array / structure can be appropriately selected from histone stem-loop arrays as disclosed in WO2012 / 019780, which disclosure is incorporated herein by reference with respect to the histone stem-loop array / histone stem-loop structure. The histone stem-loop array that can be used within the present invention can preferably be derived from formula (I) or (II) of WO2012 / 019780. According to a further preferred embodiment, the coding RNA can comprise at least one histone stem-loop array derived from at least one of the specific formulas (Ia) or (IIa) of application WO2012 / 019780.

[0241] In particularly preferred specific examples, the therapeutic RNA of the first component comprises at least one histone stem-loop array, wherein said histone stem-loop array comprises a nucleic acid sequence that is identical or at least 70%, 80%, 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.

[0242] In an embodiment, the therapeutic RNA of the first component comprises a 3'-terminal sequence element. Said 3'-terminal sequence element comprises a poly(A) sequence and a histone-stem-loop sequence, and optionally a poly(C) sequence, wherein said sequence element is located at the 3'-terminus of the RNA of the present invention.

[0243] Thus, the therapeutic RNA of the first component can comprise a 3'-terminal sequence element that is identical to SEQ ID NOs: 7 to 38 or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or a fragment or variant thereof, or consist of the same.

[0244] In various embodiments, the therapeutic RNA of the first component may comprise the 5' terminal sequence element set forth in SEQ ID NO: 5 or 6, or a fragment or variant thereof. Such 5' terminal sequence elements include, for example, the binding site for T7 RNA polymerase. Further, the first nucleotide of the 5' terminal initiation sequence may preferably comprise 2'-O-methylation, such as 2'-O-methylguanosine or 2'-O-methyladenosine.

[0245] The therapeutic RNA of the first component, preferably mRNA, may comprise a cds, 5'-UTR and / or 3'-UTR. The UTR (untranslated region) may have regulatory sequence elements or motifs that determine the turnover, stability, and / or localization of the RNA. The UTR may also have sequence elements or motifs that enhance translation. In the medical use of RNA, the translation of at least one peptide or protein of the cds is most important for the therapeutic effect. A specific combination of 3'-UTR and / or 5'-UTR may enhance the expression of an operably linked coding sequence encoding a peptide or protein as defined above. The RNA having said UTR combination may advantageously allow for rapid and transient expression of the encoded peptide or protein after administration to a subject.

[0246] Accordingly, the therapeutic RNA of the first component, preferably mRNA, may comprise a specific combination of 3'-UTR and / or 5'-UTR, resulting in (improved) translation of a therapeutic protein (e.g., a CRISPR-associated endonuclease, or an antigen), and thus resulting in protein expression in therapeutically relevant cells or tissues.

[0247] In a preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. Said 5'-UTR or 3'-UTR may be derived from a naturally occurring gene or may be synthetically engineered. In a preferred embodiment, the RNA comprises at least one cds operably linked to at least one (heterologous) 3'-UTR and / or at least one (heterologous) 5'-UTR.

[0248] In a preferred embodiment, the therapeutic RNA of the first component comprises at least one heterologous 3'-UTR.

[0249] The terms "3'-untranslated region" or "3'-UTR" or "3'-UTR element" are recognized and understood by those skilled in the art and are intended to refer to a part of the RNA located 3' (i.e., downstream) of the cds that is not translated into protein. The 3'-UTR may be part of the RNA, for example, the mRNA located between the cds and the terminal poly(A) sequence. The 3'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc.

[0250] Preferably, the therapeutic RNA of the first component, preferably mRNA, comprises a 3'-UTR derived from a gene associated with RNA having an enhanced half-life (i.e., providing a stable RNA).

[0251] In some embodiments, the 3'-UTR comprises one or more polyadenylation signals, binding sites for proteins that affect the RNA stability at the location in the cell, or binding sites for one or more miRNAs or miRNAs.

[0252] MicroRNA (or miRNA) is a non-coding RNA 19-25 nucleotides in length that binds to the 3'-UTR of a nucleic acid molecule and down-regulates gene expression by reducing the stability of the nucleic acid molecule or by inhibiting translation. For example, microRNA is known to regulate RNA, and thereby protein expression, in, for example, the liver (miR-122), heart (miR-1d, miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-194, miR-204), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133, miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126). The therapeutic RNA of the first component can include one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences can correspond to any known microRNA, such as those taught in U.S. Patent Application Publication No. 2005 / 0261218 and U.S. Patent Application Publication No. 2005 / 0059005.

[0253] Thus, to match the expression or activity of the therapeutic RNA to the desired cell type or tissue, the miRNA, or the binding site of the miRNA as defined above, can be removed from the 3'-UTR or introduced into the 3'-UTR.

[0254] In a preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, includes at least one heterologous 3'-UTR, where the at least one heterologous 3'-UTR includes a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin (referred to as "mu globin"), CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a nucleic acid sequence derived from a homolog, fragment, or variant of any one of these genes.

[0255] Particularly preferred nucleic acid sequences in this context may be derived from the published international patent application WO2019 / 077001A1, particularly claim 9 of WO2019 / 077001A1. The corresponding 3'-UTR sequences of claim 9 of WO2019 / 077001A1 are incorporated herein by reference (e.g., SEQ ID NOs: 23-34 of WO2019 / 077001A1, or fragments or variants thereof).

[0256] In other embodiments, the therapeutic RNA of the first component, preferably mRNA, comprises a 3'-UTR as described in WO2016 / 107877, and the disclosure of WO2016 / 107877 is incorporated herein by reference with respect to the 3'-UTR sequences. Suitable 3'-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016 / 107877, or fragments or variants of these sequences. In other embodiments, the therapeutic RNA comprises a 3'-UTR as described in WO2017 / 036580, and the disclosure of WO2017 / 036580 is incorporated herein by reference with respect to the 3'-UTR sequences. Suitable 3'-UTRs are SEQ ID NOs: 152-204 of WO2017 / 036580, or fragments or variants of these sequences. In other embodiments, the therapeutic RNA comprises a 3'-UTR as described in WO2016 / 022914, and the disclosure of WO2016022914 is incorporated herein by reference with respect to the 3'-UTR sequences. Particularly preferred 3'-UTRs are the nucleic acid sequences described in SEQ ID NOs: 20-36 of WO2016 / 022914, or fragments or variants of these sequences.

[0257] In a preferred embodiment, the coding RNA of the composition for use comprises at least one heterologous 5'-UTR.

[0258] The term "5'-untranslated region" or "5'-UTR" or "5'-UTR element" is recognized and understood by those skilled in the art and is intended to refer to a portion of RNA that is located 5' (i.e., "upstream") of a coding sequence that is not translated into protein. The 5'-UTR is a portion of RNA that is located on the 5' side of the coding sequence. Typically, the 5'-UTR begins at the transcription start site and ends before the start codon of the coding sequence. The 5'-UTR may contain elements, called regulatory elements, for controlling gene expression. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, and the like. The 5'-UTR may be post-transcriptionally modified, for example, by enzymatic or post-transcriptional addition of a 5'-cap structure (see above).

[0259] Preferably, the therapeutic RNA of the first component, preferably mRNA, comprises a 5'-UTR, which may be derived from a gene associated with an RNA having an enhanced half-life (i.e., providing a stable RNA).

[0260] In some embodiments, the 5'-UTR comprises one or more binding sites for proteins that affect the RNA stability at the location in the cell, or one or more miRNAs or binding sites for miRNAs (as defined above).

[0261] Accordingly, in order to tailor the expression or activity of the therapeutic RNA to a desired cell type or tissue, the miRNA or miRNA binding site defined above can be removed from the 5'-UTR or introduced into the 5'-UTR.

[0262] In a preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, comprises at least one heterologous 5'-UTR, wherein the at least one heterologous 5'-UTR is a nucleic acid sequence derived from the human and / or mouse 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUB4B, and UBQLN2, or a nucleic acid sequence derived from a homolog, fragment or variant of any one of these genes. Particularly preferred nucleic acid sequences in this context can be derived from published international application publication WO2019 / 077001A1, particularly claim 9 of WO2019 / 077001A1. The corresponding 5'-UTR sequences of claim 9 of WO2019 / 077001A1 are incorporated herein by reference (e.g., SEQ ID NOs: 1-20 of WO2019 / 077001A1, or fragments or variants thereof).

[0263] Preferably, in a preferred embodiment, the therapeutic RNA of the first component, preferably mRNA, comprises at least one cds encoding at least one peptide or protein specified herein and is operably linked to a 3'-UTR and / or 5'-UTR selected from the following combinations of 3'-UTR and / or 5'-UTR: a-1 (HSD17B4 / PSMB3), a-2 (NDUFA4 / PSMB3), a-3 (SLC7A3 / PSMB3), a-4 (NOSIP / PSMB3), a-5 (MP68 / PSMB3), b-1 (UBQLN2 / RPS9), b-3 (ASAH1 / RPS9), b-4 (HSD17B4 / RPS9), b-5 (NOSIP / COX6B1), c-1 (NDUFA4 / RPS9), c-2 (NOSIP / NDUFA4 / COX6B1), c-4 (NDUFA4 / NDUFA1), c-5 (ATP5A1 / PSMB3), d-1 (Rpl31 / PSMB3), d-2 (ATP5A1 / CASP1), d-3 (SLC7A3 / GNAS), d-4 (HSD17B4 / NDUFA1), d-5 (Slc7a3 / Ndufa1), e-1 (TUBB4b / RPS9), e-2 (RPL31 / RPS9), e-3 (MP68 / RPS9), e-4 (NOSIP / RPS9), e-5 (ATP5A1 / RPS9), e-6 (ATP5A1 / COX6B1), f-1 (ATP5A1 / GNAS), f-2 (ATP5A1 / NDUFA1), f-3 (HSD17B4 / COX6B1), f-4 (HSD17B4 / GNAS), f-5 (MP68 / COX6B1), g-1 (MP68 / NDUFA1), g-2 (NDUFA4 / CASP1), g-3 (NDUFA4 / GNAS), g-4 (NOSIP / CASP1), g-5 (RPL31 / CASP1), h-1 (RPL31 / COX6B1), h-2 (RPL31 / GNAS), h-3 (RPL31 / NDUFA1), h-4 (Slc7a3 / CASP1), h-5 (SLC7A3 / COX6B1), i-1 (SLC7A3 / RPS9), i-2 (RPL32 / ALB7), i-2 (RPL32 / ALB7), or i-3 (α-globin gene / -).

[0264] In this context, the appropriate 5'-UTR sequences defined above can be, or can be derived from, or can be derived from SEQ ID NOs: 44 to 65, or fragments or variants thereof, and the appropriate 3'-UTR sequences defined above, or SEQ ID NOs: 66 to 81, 185, 186.

[0265] In other embodiments, the therapeutic RNA of the first component, preferably mRNA, comprises a 5'-UTR as described in WO2013 / 143700, the disclosure of WO2013 / 143700 being incorporated herein by reference with respect to the 5'-UTR sequences. Particularly preferred 5'-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WO2013 / 143700, or fragments or variants of these sequences. In other embodiments, the therapeutic RNA comprises a 5'-UTR as described in WO2016 / 107877, the disclosure of WO2016 / 107877 being incorporated herein by reference with respect to the 5'-UTR sequences. Particularly preferred 5'-UTRs are nucleic acid sequences described in SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 of WO2016 / 107877, or fragments or variants of these sequences. In other embodiments, the therapeutic RNA comprises a 5'-UTR as described in WO2017 / 036580, the disclosure of WO2017 / 036580 being related to the 5'-UTR sequences and incorporated herein by reference. Particularly preferred 5'-UTRs are nucleic acid sequences described in SEQ ID NOs: 1-151 of WO2017 / 036580, or fragments or variants of these sequences. In other embodiments, the therapeutic RNA comprises a 5'-UTR as described in WO2016 / 022914, the disclosure of WO2016 / 022914 being incorporated herein by reference with respect to the 5'-UTR sequences. Particularly preferred 5'-UTRs are nucleic acid sequences described in SEQ ID NOs: 3-19 of WO2016 / 022914, or fragments or variants of these sequences.

[0266] In an embodiment, the therapeutic RNA of the first component, preferably mRNA, preferably contains the following elements in the 5' to 3' direction: A) A 5'-cap structure, preferably m7G(5')ppp(5')(2'OMeA) or m7G(5')ppp(5')(2'OMeG); B) A 5'-terminal start element preferably selected from SEQ ID NO: 5 or 6 or a fragment or variant thereof; C) For example, selected from SEQ ID NOs: 44 to 65, a 5'-UTR (preferably as specified herein); D) A ribosome binding site preferably selected from SEQ ID NO: 3 or 4, or a fragment or variant thereof; E) At least one coding sequence encoding at least one therapeutic peptide or therapeutic protein as specified herein; F) For example, selected from SEQ ID NOs: 66 to 81, a 3'-UTR preferably as specified herein; G) Optionally, a poly(A) sequence containing about 50 to about 500 adenosines; H) Optionally, a poly(C) sequence containing about 10 to about 100 cytosines; I) Optionally, a histone stem loop (sequence) preferably selected from SEQ ID NO: 1 or 2; J) Optionally, a 3'-terminal sequence consisting of SEQ ID NOs: 7 to 38.

[0267] Preferably, the therapeutic RNA of the first component, preferably mRNA, contains about 50 to about 20,000 nucleotides, or about 500 to about 10,000 nucleotides, or about 1,000 to about 10,000 nucleotides, or preferably about 1,000 to about 5,000 nucleotides.

[0268] In one embodiment, the first component (e.g., therapeutic RNA) and the second component (e.g., nucleic acid antagonist) are bound to each other.

[0269] Advantageously, such attachment can simplify the co-formulation in the carrier (see below). Ideally, the first and second components are bound to each other via non-covalent bonds, allowing for dissociation after in vivo administration. Accordingly, the invention also relates to a compound comprising a first component as defined herein and a second component as defined herein.

[0270] [Formulation of the first and / or second component] Advantageous embodiments and features regarding the formulation / complexation of at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component are described below. Further, advantageous embodiments and features regarding the formulation / complexation of at least one therapeutic RNA of the first component are described. All described embodiments and features regarding the formulation in the context of "combination" (first aspect) are equally applicable to "composition" (second aspect) or "kit or kit of parts" (third aspect).

[0271] In a preferred embodiment, the nucleic acid of the second component as defined herein and / or at least one therapeutic RNA of the first component as defined herein is complexed with, associated with, or at least partially complexed with or partially associated with one or more cationic or polycationic compounds, preferably a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, or a cationic or polycationic peptide, or any combination thereof.

[0272] In one embodiment, the nucleic acid of the second component as defined herein is bound to one or more cationic or polycationic compounds, preferably a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, or a cationic or polycationic peptide, or any combination thereof. Suitably, the therapeutic RNA of the second component complexes or associates with such cationic or polycationic compounds.

[0273] As used herein, the term "cationic or polycationic compound" is recognized and understood by those skilled in the art and refers to, for example, a positively charged molecule at a pH value in the range of about 1 to 9, a pH value in the range of about 3 to 8, a pH value in the range of about 4 to 8, a pH value in the range of about 5 to 8, more preferably a pH value in the range of about 6 to 8, even more preferably a pH value in the range of about 7 to 8, and most preferably at physiological pH, for example in the range of about 7.2 to about 7.5. Thus, a cationic component, such as a cationic peptide, a cationic protein, a cationic polymer, a cationic polysaccharide, a cationic lipid, can be any positively charged compound or polymer that is positively charged under physiological conditions. A "cationic or polycationic peptide or protein" can contain, for example, at least one positively charged amino acid selected from Arg, His, Lys, or Orn, or two or more positively charged amino acids. Thus, a "polycationic" component is also within the range of exhibiting two or more positive charges in a given state.

[0274] Cationic or polycationic compounds can particularly preferably be selected from the list of the following cationic or polycationic peptides or fragments thereof: protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-penetrating peptides (CPPs), including the following: HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived or analogous peptides, HSV VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptides, Pep-1, L-oligomers, calcitonin peptides, antennapedia-derived peptides, pAntp, pIsl, FGF, lactoferrin, transportan, Buforin-2, Bac7 15-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably, the coding RNA is complexed with one or more polycations, preferably protamine or oligofectamine, most preferably protamine.

[0275] More preferred cationic or polycationic compounds that can be used as complexing agents for the first and / or second components can include the following: cationic polysaccharides such as chitosan, polybrene, etc.; cationic lipids such as DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or polycationic polymers such as modified polyamino acids such as β-amino acid polymers or reverse polyamides, etc., modified polyethylene such as PVP, etc., modified acrylates such as pDMAEMA, etc., modified amidoamines such as pAMAM, etc., modified poly-β-amino esters (PBAE) such as diamine-terminated modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymer, etc., dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, etc., polyimines such as PEI, poly(propyleneimine), etc., polyallylamine, sugar backbone-based polymers such as cyclodextrin-based polymers, dextran-based polymers, etc., silane backbone-based polymers such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks (selected from cationic polymers such as those described above) and one or more hydrophilic or hydrophobic blocks (such as polyethylene glycol); etc.

[0276] Preferred cationic or polycationic proteins or peptides that can be used for complexing the first component and / or the second component can be derived from the formula (Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x of patent application WO2009 / 030481 or WO2011 / 026641, and the disclosures of WO2009 / 030481 or WO2011 / 026641 related thereto are incorporated herein by reference.

[0277] In various embodiments, one or more cationic or polycationic peptides of the first and / or second component are selected from SEQ ID NOs: 39-43, or any combination thereof.

[0278] Thus, in a preferred embodiment, at least one antagonist of the second component, preferably a nucleic acid, is complexed with, or associates with, or is at least partially complexed with, or partially associates with one or more cationic or polycationic peptides selected from SEQ ID NOs: 39-43, or any combination thereof.

[0279] Thus, in a preferred embodiment, at least one therapeutic RNA of the first component, preferably mRNA, is complexed with, or associates with, or is at least partially complexed with, or partially associates with one or more cationic or polycationic peptides selected from SEQ ID NOs: 39-43, or any combination thereof.

[0280] In an embodiment, the nucleic acid of the second component as defined herein is complexed with, or associates with, or is at least partially complexed with, or partially associates with one or more cationic polymers or polycationic polymers.

[0281] In an embodiment, at least one therapeutic RNA of the first component, preferably mRNA, is complexed with, or associates with, or is at least partially complexed with, or partially associates with one or more cationic or polycationic polymers.

[0282] Thus, in an embodiment, the first and / or second component comprises at least one polymeric carrier.

[0283] As used herein, the term "polymer carrier" is recognized and understood by those skilled in the art and is intended to refer to, for example, a compound that facilitates the transport and / or complexation of another compound (e.g., a first, second component). A polymer carrier is typically a carrier formed from a polymer. A polymer carrier can associate with its payload (e.g., RNA) by covalent or non-covalent interactions. Polymers, such as copolymers, can be based on different subunits.

[0284] Suitable polymeric carriers in that context include, for example, the following: polyacrylate, polyalkylanoacrylate, polylactide, polylactide - polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PEGylated PLL and polyethyleneimine (PEI), dithiobis(succinimidyl propionate) (DSP), dimethyl - 3,3’ - dithiobispropionimidate (DTBP), poly(ethyleneimine) biscarbamate (PEIC), poly(L - lysine), histidine - modified PLL, poly(N - vinylpyrrolidone) (PVP), poly(propyleneimine) (PPI), poly(amidoamine) (PAMAM), poly(amidoethyleneimine) (SS - PAEI), triethylenetetramine (TETA), poly(β - amino ester), poly(4 - hydroxy - L - proine ester) (PHP), poly(allylamine), poly(α - [4 - aminobutyl] - L - glycolic acid) (PAGA), poly(D,L - lactic - co - glycolic acid) (PLGA), poly(N - ethyl - 4 - vinylpyridinium bromide), poly(phosphazene) (PPZ), poly(phosphoester) (PPE), poly(phosphoamidate) (PPA), poly(N - 2 - hydroxypropylmethacrylamide) (pHPMA), poly(2 - (dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2 - aminoethylpropylene phosphate) (PPE_EA), galactosylated chitosan, N - dodecylated chitosan, histone, collagen and dextran - spermine. In one embodiment, the polymer may be an inert polymer such as PEG, but is not limited thereto. In one embodiment, the polymer may be a cationic polymer such as PEI, PLL, TETA, poly(allylamine), poly(N - ethyl - 4 - vinylpyridinium bromide), pHPMA and pDMAEMA, but is not limited thereto. In one embodiment, the polymer may be a biodegradable PEI such as DSP, DTBP and PEIC, but is not limited thereto.In one embodiment, the polymer may be biodegradable such as, but not limited to, histone-modified PLL, SS-PAEI, poly(β-amino ester), PHP, PAGA, PLGA, PPZ, PPE, PPA, and PPE-EA.

[0285] A suitable polymer carrier can be a polymer carrier formed by a disulfide-crosslinked cationic compound. The disulfide-crosslinked cationic compounds may be the same as or different from each other. The polymer carrier can also contain additional components (such as lipidoid compounds). The polymer carriers used in accordance with the present invention can include a mixture of cationic peptides, proteins, or polymers crosslinked by disulfide bonds (via -SH groups), and optionally additional components as defined herein.

[0286] In this context, polymer carriers according to the formulas (Ia) {(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa’)x(Cys)y} and (Ib) Cys{(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys of published International Patent Application No. WO 2012 / 013326 are preferred and are incorporated herein by reference.

[0287] In an embodiment, the polymer carrier used to complex at least one coding RNA can be derived from a polymer carrier molecule according to the formula (L-P 1 -S-[S-P 2 -S] n -S-P 3 -L) of published PCT application WO2011 / 026641, the disclosure of WO2011 / 026641 related thereto being incorporated herein by reference.

[0288] In an embodiment, the polymer carrier compound is formed by, contains, or consists of a peptide having CysArg12Cys (SEQ ID NO: 39) or CysArg12 (SEQ ID NO: 40) or TrpArg12Cys (SEQ ID NO: 41) as an element. In other embodiments, the polymer carrier compound is formed by, contains, or consists of a peptide element according to SEQ ID NO: 42 or 43.

[0289] In a particularly preferred specific example, this polymer carrier complex consists of an (R 12 C)-(R 12 C) dimer, a (WR 12 C)-(WR 12 C) dimer, or a (CR 12 )-(CR 12 C)-(CR 12 ) trimer, and the individual peptide elements of the dimer (e.g., (WR12C)) or trimer (e.g., (CR12)) are bonded via -SH groups.

[0290] In a preferred embodiment, the cationic or polycationic polymer of the first and / or second component is a polyethylene glycol / peptide polymer containing HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (peptide monomer of SEQ ID NO: 42), and / or a polyethylene glycol / peptide polymer containing HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-OH (peptide monomer of SEQ ID NO: 43).

[0291] In embodiments, the first and / or second component is complexed or associated with a polymer carrier, optionally with at least one lipid or lipidoid, as described in International Publication Nos. WO 2017 / 212008A1, WO 2017 / 212006A1, WO 2017 / 212007A1, and WO 2017 / 212009A1, the contents of which are incorporated herein by reference.

[0292] In a particularly preferred embodiment, the polymer carrier (of the first and / or second component) is a peptide polymer, preferably a polyethylene glycol / peptide polymer as defined above, and a lipid, preferably a lipidoid.

[0293] A lipidoid (or lipid oid) is a lipid-like compound, i.e., an amphiphilic compound having lipid-like physical properties. A lipidoid preferably contains two or more cationic nitrogen atoms and at least two lipophilic tails. In contrast to many conventional cationic lipids, a lipidoid may not contain a hydrolysable linking group, particularly a linking group containing a hydrolysable ester, amide or carbamate group. The cationic nitrogen atoms of a lipidoid may be cationizable or permanently cationic, or both types of cationic nitrogen may be present in the compound. In the context of the present invention, the term lipid is considered to include lipidoids as well.

[0294] In some embodiments of the present invention, the lipidoid can contain a PEG moiety.

[0295] Suitably, the lipidoid is cationic, which means it is cationic or permanently cationic. In one embodiment, the lipidoid is cationizable, i.e., it contains one or more cationizable nitrogen atoms but does not contain permanently cationic nitrogen atoms. In another embodiment, at least one of the cationic nitrogen atoms of the lipidoid is permanently cationic. Optionally, the lipidoid contains two permanently cationic nitrogen atoms, three permanently cationic nitrogen atoms, or even four or more permanently cationic nitrogen atoms.

[0296] As a specific example, the lipidoid can be selected from any of the lipidoids provided in the table on pages 50 - 54 of the published PCT patent application WO2017 / 212009A1, and the specific lipidoids provided in the said table, and the related specific disclosures are incorporated herein by reference.

[0297] In a preferred embodiment, the lipidoid is selected from the following: 3-C12-OH, 3-C12-OH-cat, 3-C12-amide, 3-C12-amide monomethyl, 3-C12-amide dimethyl, RevPEG(10)-3-C12-OH, RevPEG(10)-DLin-pAbenzoic, 3C12 amide-TMA cat., 3C12 amide-DMA, 3C12 amide-NH2, 3C12 amide-OH, 3C12Ester-OH, 3C12 Ester-amin, 3C12Ester-DMA, 2C12Amid-DMA, 3C12-lin-amid-DMA, 2C12-sperm-amid-DMA or 3C12-sperm-amid-DMA (see the table on pages 50 - 54 of the published PCT patent application WO2017 / 212009A1). Particularly preferred lipidoids in the context of the present invention are 3-C12-OH or 3-C12-OH-cat.

[0298] In a preferred embodiment, a peptide polymer comprising a lipidoid as specified above complexes at least one therapeutic RNA of the first component and / or at least one antagonist (e.g., nucleic acid) of the second component to form a complex having an N / P ratio of about 0.1 to about 20, or about 0.2 to about 15, or about 2 to about 15, or about 2 to about 12, where the N / P ratio is defined as the molar ratio of the nitrogen atoms of the basic groups of the cationic peptide or polymer to the phosphate groups of the nucleic acid. In this context, the disclosure of published International Application Publication No. WO 2017 / 212009A1, particularly the disclosure of claims 1 to 10 of International Publication No. WO 2017 / 212009A1, and the specific disclosures related thereto are incorporated herein by reference.

[0299] In certain embodiments, at least one therapeutic RNA of the first component, preferably mRNA, complexes or associates with a polymer carrier, preferably a polyethylene glycol / peptide polymer as defined above, and a lipidoid, preferably 3-C12-OH and / or 3-C12-OH-cat.

[0300] In certain embodiments, at least one antagonist of the second component, preferably a nucleic acid, complexes or associates with a polymer carrier, preferably a polyethylene glycol / peptide polymer as defined above, and a lipidoid, preferably 3-C12-OH and / or 3-C12-OH-cat.

[0301] Further suitable lipidoids can be derived from published PCT patent application WO2010 / 053572. In particular, lipidoids derived from claims 1 to 297 of published International Application Publication No. WO 2010 / 053572 can be used in the context of the present invention, for example, incorporated into a peptide polymer as described herein, or incorporated into lipid nanoparticles (as described below). Accordingly, the disclosure of claims 1 to 297 of published International Application Publication No. WO 2010 / 053572, and the specific descriptions related thereto are incorporated herein by reference.

[0302] In a preferred embodiment, at least one therapeutic RNA, preferably mRNA, of the first compound is complexed with, partially complexed with, encapsulated by, partially encapsulated by, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes.

[0303] In a preferred embodiment, at least one antagonist of the second compound, preferably a nucleic acid, is complexed with, partially complexed with, encapsulated by, partially encapsulated by, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes.

[0304] Liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes - incorporating a therapeutic RNA of a first compound or an antagonist (e.g., nucleic acid) of a second compound - can be located completely or partially within the internal space of the liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes, within the membrane, or associated with the outer surface of the membrane. The incorporation of the therapeutic RNA of the first compound or the antagonist of the second compound is also referred to herein as "encapsulation", where the defined therapeutic RNA or antagonist (e.g., nucleic acid) is completely contained within the internal space of the liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes. The purpose of incorporating the first and / or second components into liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes is to protect the components from, for example, enzymes or chemicals that degrade the therapeutic RNA and / or an environment that may contain systems or receptors that cause rapid excretion of the therapeutic RNA. Further, incorporating the first and / or second components into liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes can facilitate the uptake of the RNA and thus enhance their therapeutic effects.

[0305] In this context, the terms "complexed" or "associated" refer to an essentially stable combination of a therapeutic RNA of a first component or an antagonist (e.g., nucleic acid) of a second component, as defined herein, with one or more lipids into a larger complex or assembly without covalent bonds.

[0306] The term "lipid nanoparticles", also referred to as "LNP", is not limited to any particular morphology and includes, for example, any morphology produced when a cationic lipid and optionally one or more additional lipids are combined in an aqueous environment and / or in the presence of RNA. For example, liposomes, lipid complexes, lipoplexes, etc. are within the scope of LNP.

[0307] Liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes can have diameters in the hundreds of nanometers and can include a series of concentric bilayer membranes separated by narrow aqueous compartments, small unilamellar vesicles (SUV) that can have diameters of less than 50 nm, and multilamellar vesicles (MLV) that can include large unilamellar vesicles (LUV) that can have diameters between 50 nm and 500 nm, among other possible sizes, but are not limited thereto.

[0308] The LNP of the present invention is suitably characterized as a microscopic vesicle having an internal aqueous space isolated from the external medium by one or more bilayer membranes. The bilayer membrane of the LNP is typically formed by amphiphilic molecules such as lipids of synthetic or natural origin that contain spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of a liposome can also be formed by amphiphilic polymers and surfactants (e.g., polymelrosomes, niosomes, etc.).

[0309] Thus, in a preferred embodiment, at least one therapeutic RNA of the first component and / or at least one antagonist (e.g., nucleic acid) of the second component is complexed with one or more lipids, thereby forming lipid nanoparticles (LNP).

[0310] LNPs typically comprise at least one cationic lipid and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., PEGylated lipids). At least one therapeutic RNA / at least one antagonist (e.g., nucleic acid) as defined herein may be encapsulated in the aqueous space enclosed by the lipid portion of the LNP, or a part or all of the lipid portion of the LNP. At least one therapeutic RNA / at least one antagonist (e.g., nucleic acid) or a portion thereof may also associate and complex with the LNP. The LNP may comprise any lipid capable of forming particles to which nucleic acids attach or in which one or more nucleic acids are encapsulated. Preferably, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

[0311] The cationic lipid of the LNP may be cationizable, i.e., it is protonated as the pH decreases below the pK of the ionizable substituent of the lipid, but becomes gradually electrically neutral at higher pH values. At pH values below the pK, the lipid can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises zwitterionic lipids that assume a positive charge upon a decrease in pH.

[0312] Such lipids include, but are not limited to, the following: DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylammonium propane chloride salt (DLin-TMA.Cl), ICE (imidazole-based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-dilinoleoyl-3-trimethylammonium propane chloride salt (DLin-TAP.Cl), 1,2-dilinoleoyl-oxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyl-oxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butanoate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1,1´-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-doundecyl-4,7,10,13-tetraazhexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,(31 - tetraen - 19 - yloxy)-N,N - dimethylbutan - 1 - amine (MC4 ether), LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2 - dioleoyl - sn - phosphoethanolamine (DOPE) from GIBCO / BRL, Gr and Isl, N.Y.); LIPOFECTAMINE® (a commercially available cationic liposome containing N - (1 - (2,3 - dioleyloxy)propyl)-N - (2 - (sperminecarboxamido)ethyl)-N,N - dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and, TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.) or any combination of the above. Further suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication WO2010 / 053572 (in particular, CI 2 - 200 described in paragraph

[0225] ) and WO2012 / 170930, which are hereby incorporated by reference herein, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1).

[0313] In an embodiment, the cationic lipid may be an amino lipid.

[0314] Representative amino lipids include, but are not limited to: 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3 morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3 dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyl-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).

[0315] In one embodiment, at least one therapeutic RNA as defined herein / antagonist (e.g., nucleic acid) as defined herein can be formulated in an amino alcohol lipidoid. The amino alcohol lipids that can be used in the present invention can be prepared by the method described in U.S. Patent No. 8,450,298, which is hereby incorporated by reference in its entirety. Suitable (ionizable) lipids can also be the compounds disclosed in Tables 1, 2, and 3 and defined in claims 1-24 of published International Application Publication No. 2017 / 075531A1, the specific disclosure of which is hereby incorporated by reference herein.

[0316] In other embodiments, suitable lipids may be selected from International Publication WO2015 / 074085A1 (i.e., ATX - 001 to ATX - 032, or the compounds specified in claims 1 - 26), U.S. Patent Application No. 61 / 905,724, 15 / 614,499 or U.S. Patent Nos. 9,593,077 and 9,567,296, which are incorporated herein by reference.

[0317] In other embodiments, suitable cationic lipids may be selected from Published International Application Publication No. 2017 / 117530A1 (i.e., lipid 13, 14, 15, 16, 17, 18, 19, 20, or the compounds specified in the claims), and the specific description is incorporated herein by reference.

[0318] In a preferred embodiment, the ionizable lipid / cationic lipid may also be selected from the lipids disclosed in Published International Application No. 2018 / 078053A1 (i.e., the lipids derived from Formulas I, II, and III of International Publication No. 2018 / 078053A1, or the lipids specified in claims 1 - 12 of International Publication No. 2018 / 078053A1), and the specific disclosure of WO2018 / 078053A1 is incorporated herein by reference. In that context, the lipids disclosed in Table 7 of WO2018 / 078053A1 (e.g., the lipids derived from Formulas I - 1 to I - 41) and the lipids disclosed in Table 8 of WO2018 / 078053A1 (e.g., the lipids derived from Formulas II - 1 to II - 36) can be appropriately used in the context of the present invention. Accordingly, Formulas I - 1 to I - 41 and Formulas II - 1 to II - 36 of WO2018 / 078053A1, and the specific disclosures related thereto are incorporated herein by reference.

[0319] In a preferred embodiment, the cationic lipid may be derived from Formula III of Published International Application Publication No. 2018 / 078053A1. Accordingly, Formula III of WO2018 / 078053A1, and the specific disclosures related thereto are incorporated herein by reference.

[0320] In particularly preferred specific examples, at least one therapeutic RNA as defined herein / an antagonist (e.g., a nucleic acid) as defined herein is complexed with one or more lipids, thereby forming an LNP, wherein the cationic lipid of the LNP is selected from Structures III-1 to III-36 in Table 9 of Published International Patent Application Publication No. WO 2018 / 078053A1. Accordingly, the formulas III-1 to III-36 of WO2018 / 078053A1, and the specific disclosures related thereto, are incorporated herein by reference.

[0321] In a particularly preferred specific example, at least one therapeutic RNA as defined herein / an antagonist (e.g., a nucleic acid) as defined herein is complexed with one or more lipids, thereby forming an LNP, wherein the LNP comprises the following cationic lipids:

[0322]

Chemical formula

[0323] In certain embodiments, the cationic lipid (e.g., III-3) is present in the LNP in an amount of about 30 to about 95 mol% relative to the total lipid content of the LNP. When two or more cationic lipids are incorporated within the LNP, such percentages apply to the combined cationic lipids.

[0324] Other suitable (cationic or ionizable) lipids are disclosed in the following: published patent applications WO2009 / 086558, WO2010 / 048536, WO2010 / 054406, WO2010 / 088537, WO2011 / 129709, WO2011 / 063468, US2011 / 0256175, US2012 / 0027801, WO2016 / 118725, WO2017 / 070613, WO2017 / 09823, WO2012 / 040184, WO2011 / 153120, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, WO2008 / 103276, WO 2013 / 086354, and U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and U.S. Patent Publications US2010 / 0036115, US2012 / 0202871, US2013 / 0064894, US2013 / 0129785, US2013 / 0150625, US20130178541, US2013 / 0225836, US2014 / 0039032 and WO2017 / 112865.In that context, WO2009 / 086558, WO2010 / 048536, WO2010 / 088537, WO2010 / 129709, WO2011 / 153493, WO2011 / 0254468, US2011 / 0256175, US2012 / 0027803, WO2016 / 118725, WO2017 / 070620, WO2017 / 099823, WO2012 / 040184, WO2011 / 153120, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, WO2008 / 103276, WO 2013 / 086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,256, and 8,569,256, as well as US2010 / 0036115, US2012 / 0202871, US2013 / 0064894, US2013 / 0129785, US2013 / 0129785, US2013 / 0150625, US20130178541, US2013 / 0225836, and US2014 / 0039032 and WO2017 / 112865 relate to (cationic) lipids particularly suitable for LNPs and are hereby incorporated by reference.

[0325] The LNP can comprise two or more (different) cationic lipids. The cationic lipids can be selected to contribute various advantageous properties. For example, cationic lipids having different properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, toxicity, or immunostimulation can be used in the LNP.

[0326] The in vivo properties and behavior of LNPs can be modified to impart steric stabilization by coating the LNP surface with a hydrophilic polymer coating, such as polyethylene glycol (PEG). Additionally, LNPs can be used for specific targeting by attaching ligands (such as antibodies, peptides, and carbohydrates) to their surface or the termini of attached PEG chains (e.g., via PEGylated lipids or PEGylated cholesterol).

[0327] In some embodiments, such PEG chains can be used to bind an antagonist of the invention.

[0328] In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule that includes both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that includes both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include, for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-sDMG).

[0329] In various embodiments, the LNP comprises a stabilizing lipid that is a polyethylene glycol lipid (PEGylated lipid). Suitable polyethylene glycol lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, PEG-DMG, PEG-DSG, PEG-DSPE, PEG-DOMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DMG), e.g., 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or, PEG dialkoxypropylcarbamate, e.g., ω-(polyethoxy)ethyl-(2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0330] In a preferred embodiment, the PEGylated lipid preferably derives from formula (IV) of published International Application Publication No. WO2018 / 078053A1. Accordingly, the PEGylated lipids derived from formula (IV) of published International Application Publication No. WO2018 / 078053A1, and the respective descriptions related thereto, are incorporated herein by reference.

[0331] In particularly preferred embodiments, the therapeutic RNA of the first component and / or at least one antagonist of the second component are complexed with one or more lipids, thereby forming an LNP, where the LNP comprises a PEGylated lipid, where the PEG lipid preferably derives from formula (IVa) of published International Application Publication WO2018 / 078053A1. Accordingly, the PEGylated lipids derived from formula (IVa) of published International Application Publication No. WO2018 / 078053A1, and the respective descriptions related thereto, are incorporated herein by reference.

[0332] In a particularly preferred embodiment, the PEG lipid is of formula (IVa):

[0333]

Chemical formula

[0334] where n has an average value of from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In the most preferred embodiment, n is about 49.

[0335] Further examples of PEG lipids appropriate in that context are provided in US2015 / 0376115A1 and WO2015 / 199952, each of which is incorporated herein by reference in its entirety.

[0336] In some embodiments, the LNP contains less than about 3, 2, or 1 mole percent of PEG or PEGylated lipid, based on the total moles of lipid in the LNP. In further embodiments, the LNP contains from about 0.1% to about 20% PEGylated lipid on a molar basis. In preferred embodiments, the LNP contains from about 1.0% to about 2.0% PEGylated lipid on a molar basis. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0337] In preferred embodiments, the LNP contains one or more additional lipids (such as neutral lipids and / or one or more steroids or steroid analogs) that stabilize the formation of the particles during their formation or during a manufacturing process.

[0338] In preferred embodiments, the LNP contains one or more neutral lipids and / or one or more steroids or steroid analogs.

[0339] Suitable stabilizing lipids include neutral lipids and anionic lipids. The term "neutral lipid" refers to any one of many lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0340] In embodiments, the LNP comprises one or more neutral lipids, where the neutral lipid is selected from the group comprising: distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-transPE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (transDOPE) or mixtures thereof.

[0341] In some embodiments, the LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1. In a preferred embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The molar ratio of the cationic lipid to DSPC can range from about 2:1 to 8:1. In a preferred embodiment, the steroid is cholesterol. The molar ratio of the cationic lipid to cholesterol can range from about 2:1 to 1:1. In some embodiments, cholesterol may be PEGylated.

[0342] In a particularly preferred embodiment, the lipid compound is a lipid compound or is derived from Formula III, preferably III-3, the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is a compound of Formula (IVa).

[0343] In a preferred embodiment, the liposomes, lipid nanoparticles, lipoplexes, and / or nanoliposomes preferably comprise or consist of: (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one aggregation-reducing lipid, wherein preferably (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

[0344] In a specific embodiment, at least one therapeutic RNA of the first component and / or at least one antagonist (e.g., nucleic acid) of the second component is complexed with one or more lipids, thereby forming an LNP, where the LNP (i) at least one cationic lipid as defined herein, preferably lipid III-3; (ii) at least one neutral lipid as defined herein, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog as defined herein, preferably cholesterol; and, (iv) at least one PEG-lipid as defined herein, e.g., PEG-DMG or PEG-cDMA, preferably the PEGylated lipid of Formula (IVa) comprises, wherein preferably (i)-(iv) are in a molar ratio of about 20-60% cationic lipid; 5-25% neutral lipid; 25-55% sterol; 0.5-15% PEG-lipid.

[0345] In certain preferred embodiments, at least one therapeutic RNA of the first component and / or at least one antagonist (e.g., nucleic acid) of the second component are complexed with one or more lipids, thereby forming an LNP, where the LNP has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e., the ratio (mol%) of solubilized in ethanol, cationic lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid (preferably PEG-lipid of formula (IVa) (n = 49))).

[0346] In various embodiments, the LNP as defined herein has an average diameter of about 50 nm to about 200 nm, about 50 nm to about 150 nm, or about 50 nm to about 100 nm. As used herein, the average diameter can be represented by the z-average determined by dynamic light scattering, as is generally known in the art. The polydispersity index (PDI) of the LNP is preferably in the range of 0.1 to 0.5. In certain embodiments, the PDI is less than 0.2. Typically, the PDI is determined by dynamic light scattering as is generally known in the art.

[0347] In a preferred aspect, the administration of the combination, preferably the administration of the first and second components, is essentially simultaneous.

[0348] "Simultaneously" in this context is to be understood as meaning that the administration of the first and second components of the combination can occur at the same time, rather than in a staggered, well-timed manner. The co-administration may be via the same administration site / administration route or different administration sites / administration routes, as further outlined below.

[0349] In other preferred embodiments, the administration of the combination, preferably the administration of the first and second components, is continuous.

[0350] In this context, "continuous" is to be understood as meaning that the administration of the first and second components of the combination can occur in a timely manner, with staggered timing, rather than simultaneously. Said "continuous" administration may be at the same administration site or at different administration sites, as further outlined below.

[0351] In a preferred embodiment, the administration of the combination, i.e., the administration of the first component and / or the second component (continuous or simultaneous), is carried out two or more times, for example, once or more per day, once or more per week, once or more per month. Advantageously, the combination of the present invention is suitable for repeated administration, for example, for chronic administration.

[0352] The present combination can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramedullary, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, intraocular, intravitreal, subretinal, intratumoral.

[0353] In a particularly preferred embodiment, the administration of the present combination, particularly the administration of the first component and / or the second component (continuous or simultaneous), is carried out intravenously. In a specific embodiment, the present combination is administered intravenously as a chronic treatment (e.g., two or more times, for example, once or more per day, once or more per week, once or more per month, or two or more times per month).

[0354] In a particularly preferred embodiment, the present combination is characterized by the following features: (I) at least one first component as defined herein, preferably a therapeutic peptide or a therapeutic protein, such as an mRNA encoding an antibody, an enzyme, an antigen, where optionally, said mRNA does not contain modified nucleotides, where said mRNA contains a Cap1 structure (preferably obtainable by co-transcriptional capping), where said first component is formulated in lipid nanoparticles or a polyethylene glycol / peptide polymer, the first component (II) At least one second component as defined herein, preferably a single-stranded RNA oligonucleotide comprising at least one 2'-O-methylated RNA nucleotide, preferably comprising a nucleic acid sequence according to formula I, wherein said second component is formulated in a lipid nanoparticle or in a polyethylene glycol / peptide polymer, said oligonucleotide.

[0355] In some embodiments, administration of the present combination to a cell, tissue, or organism results in an increase in expression, for example compared to administration of the corresponding first component alone. In particular, a decrease in (innate) immune activation promotes translation of the first component.

[0356] [Composition] In a second aspect, the present invention provides a composition comprising a first component as defined herein and a second component as defined herein.

[0357] In a preferred embodiment, the pharmaceutical composition comprises or consists of: (i) at least one therapeutic RNA; (ii) at least one antagonist of at least one RNA-sensing pattern recognition receptor, and, Optionally, at least one pharmaceutically acceptable carrier.

[0358] Preferably, at least one therapeutic RNA is as described as the "first component" in the context of the present combination, and at least one antagonist is as described as the "second component" in the context of the present combination. Accordingly, the above embodiments regarding the first component of the present combination (in the context of the first aspect) are also applicable to at least one therapeutic RNA of the composition. Further, the above embodiments regarding the second component of the present combination (in the context of the first aspect) are also applicable to at least one antagonist of at least one RNA-sensing pattern recognition receptor of the composition.

[0359] In a preferred embodiment, the pharmaceutical composition of the second aspect consists of, or comprises, the combination as defined in the context of the first aspect and optionally at least one pharmaceutically acceptable carrier.

[0360] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" preferably includes a liquid or non-liquid base for the first component and / or the second component. When the first and / or second component is provided in liquid form, the carrier may be water, such as pyrogen-free water, isotonic saline or a buffered (aqueous) solution, such as a phosphate, citrate, etc. buffer solution. A water or preferably a buffer, more preferably an aqueous buffer, containing a sodium salt, preferably at least 50 mM sodium salt, a calcium salt, preferably at least 0.01 mM calcium salt, and optionally a potassium salt, preferably at least 3 mM potassium salt, can be used. According to a preferred embodiment, the sodium salt, calcium salt, and optionally potassium salt can be present in the form of their halides, such as chlorides, iodides, or bromides, or in the form of their hydroxides, carbonates, bicarbonates, or sulfates, etc. Examples of sodium salts include NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of optional potassium salts include KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2. In particular, a suitable pharmaceutically acceptable carrier refers to a substance that does not interfere with the effectiveness of the first and / or second component, combination or component as defined herein and is compatible with biological systems such as cells, cell cultures, tissues, or organisms.

[0361] Further advantageous embodiments and features of the pharmaceutical compositions of the present invention are described below. In particular, the embodiments and features described in the context of the pharmaceutical compositions may similarly be applicable to the combination of the first aspect and / or the kit or parts kit of the third aspect.

[0362] Accordingly, the pharmaceutical composition comprises or consists of the following: (i) at least one therapeutic RNA, wherein the at least one therapeutic RNA is the "first component" as defined in the context of the first aspect; (ii) at least one antagonist of at least one RNA-sensing pattern recognition receptor, wherein the at least one antagonist is the "second component" as defined in the context of the first aspect; Optionally, at least one pharmaceutically acceptable carrier, preferably the pharmaceutically acceptable carrier as defined above.

[0363] In a preferred embodiment, the pharmaceutical composition comprises or consists of the following: (i) at least one therapeutic RNA, wherein the at least one therapeutic RNA is the "first component"; (ii) at least one antagonist of at least one RNA-sensing pattern recognition receptor, wherein the at least one antagonist is the "second component", preferably a nucleic acid.

[0364] The composition suitably contains a safe and effective amount of the therapeutic RNA as specified herein. As used herein, "safe and effective amount" means an amount of the therapeutic RNA, preferably mRNA, sufficient to effect the expression and / or activity of the encoded protein after administration. At the same time, the "safe and effective amount" is small enough to avoid serious side effects caused by the administration of the therapeutic RNA.

[0365] Furthermore, the composition suitably comprises a safe and effective amount of at least one antagonist of at least one RNA-sensing pattern recognition receptor, preferably a nucleic acid as specified herein. As used herein, "safe and effective amount" means an amount of antagonist (preferably a nucleic acid) sufficient to cause antagonism of at least one RNA-sensing pattern recognition receptor after administration. At the same time, the "safe and effective amount" is small enough to avoid serious side effects caused by administration of the antagonist.

[0366] The "safe and effective amounts" of the first and second components of the composition further vary in relation to the particular condition being treated, as well as the age and physical condition of the patient being treated, the severity of the condition, the duration of treatment, the nature of any concomitant treatments, the particular pharmaceutically acceptable carrier used, and the like. Further, the "safe and effective amounts" of the first and second components as described herein may depend on the route of administration (e.g., intravenous, intramuscular), the administration device (needle injection, injection device), and / or complex formation / formulation (e.g., RNA conjugated to a polymeric carrier or LNP). Further, the "safe and effective amount" of the composition may depend on the condition of the subject being treated (e.g., infant, immunocompromised human subject, etc.).

[0367] In the context of the present invention, "composition" refers to any kind of composition in which specific components (e.g., a first component as defined herein, e.g., mRNA, and / or a second component as defined herein, e.g., a nucleic acid) can be incorporated, optionally together with further components, usually together with at least one pharmaceutically acceptable carrier or excipient. The composition may be a dry composition such as a powder or granule, or a solid unit such as a lyophilized form. The composition may be liquid, and each component may be incorporated in a dissolved or dispersed (e.g., suspended or emulsified) form independently.

[0368] As used herein, the terms "subject", "patient" or "individual" generally include humans and non-human animals, preferably mammals (including chimeric and transgenic animals as well as disease models). Subjects to whom administration of a composition, preferably a pharmaceutical composition, is intended include humans and / or other primates; mammals including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, etc.; and / or commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys, but are not limited thereto. Preferably, the term "subject" refers to non-human primates or humans, most preferably humans.

[0369] In a preferred embodiment, a "subject in need of treatment" or "subject in need of therapy" in the context of the present invention is a human subject.

[0370] In an embodiment, the composition may comprise a plurality or at least two or more therapeutic RNA species as defined above, wherein each therapeutic RNA species, e.g., each mRNA species, may encode a different therapeutic peptide or protein as defined.

[0371] In an embodiment, the composition comprises two or more or a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, of the first components as defined above.

[0372] As used herein, the term "RNA species" is not intended to refer to only one single molecule. The term "RNA species" must be understood as an aggregate of essentially identical RNA molecules, where each of the RNA molecules of the RNA aggregate, in other words, each of the molecules of the RNA species, encodes the same therapeutic protein (in embodiments where the therapeutic RNA is a coding RNA) having essentially the same nucleic acid sequence. However, the RNA molecules of the RNA aggregate may differ in length or quality that can be caused by enzymatic or chemical manufacturing processes.

[0373] In an embodiment, the composition comprises two or more or a plurality of different therapeutic RNA species of the first component, wherein the two or more or plurality of different therapeutic RNA species are each selected from coding RNA species encoding different proteins.

[0374] In an embodiment, the composition comprises one or more or a plurality of different therapeutic RNA species of the first component, wherein at least one of the two or more or plurality of different therapeutic RNA species is selected from coding RNA species (e.g., mRNA encoding a CRISPR-associated endonuclease), and at least one is selected from non-coding RNA species (e.g., guide RNA).

[0375] In an embodiment, the composition comprises two or more or a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, different antagonists of the second component as defined above, preferably nucleic acid species.

[0376] As used herein, the term "nucleic acid species" is not intended to refer to only a single nucleic acid molecule. The term "nucleic acid species" in the context of the second component must be understood as an aggregate of essentially identical nucleic acid molecules, and each of the nucleic acid molecules of such an aggregate has an essentially identical nucleic acid sequence.

[0377] In a preferred embodiment, the composition comprises a therapeutic RNA of a first component, preferably mRNA, and an antagonist of a second component, preferably a nucleic acid, wherein the first component and / or the second component is complexed or associated with, or at least partially complexed or associated with, one or more cationic or polycationic compounds, preferably a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, or a cationic or polycationic peptide, or any combination thereof. Complexation / association to a carrier as defined herein ( "formulation") facilitates the uptake of the therapeutic RNA and / or antagonist into cells.

[0378] As used herein, the term "cationic or polycationic compound" is recognized and understood by those skilled in the art and refers to, for example, a positively charged molecule at a pH value in the range of about 1 to 9, a pH in the range of about 3 to 8, a pH in the range of about 4 to 8, a pH value in the range of about 5 to 8, more preferably a pH value in the range of about 6 to 8, even more preferably a pH value in the range of about 7 to 8, and most preferably a physiological pH, such as in the range of about 7.2 to about 7.5. Thus, cationic components, such as cationic peptides, cationic proteins, cationic polymers, cationic polysaccharides, cationic lipids (including lipidoids), may be any positively charged compound or a polymer that is positively charged under physiological conditions. A "cationic or polycationic peptide or protein" may contain at least one positively charged amino acid selected from, for example, Arg, His, Lys or Orn, or two or more positively charged amino acids. Thus, a "polycationic" component is also within the range of exhibiting two or more positive charges in a given state.

[0379] Particularly preferred cationic or polycationic compounds in this context can be selected from the list of the following cationic or polycationic peptides or fragments thereof: protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-penetrating peptides (CPPs), including the following: HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived or analog peptides, HSV VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptides, Pep-1, L-oligomers, calcitonin peptides, antennapedia-derived peptides, pAntp, pIsl, FGF, lactoferrin, transportan, Buforin-2, Bac7 15-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones.

[0380] More preferred cationic or polycationic compounds that can be used as transfection or complexing agents include the following: cationic lipids such as DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or polycationic polymers such as modified polyamino acids such as β-amino acid polymers or reverse polyamides, etc., modified polyethylene such as PVP, etc., modified acrylates such as pDMAEMA, etc., modified amidoamines such as pAMAM, etc., modified poly-β-amino esters (PBAE) such as diamine-terminated modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymer, etc., dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, etc., polyimines such as PEI, poly(propyleneimine), etc., polyallylamine, sugar backbone-based polymers such as cyclodextrin-based polymers, dextran-based polymers, etc., silane backbone-based polymers such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks (selected from cationic polymers such as those described above) and one or more hydrophilic or hydrophobic blocks (such as polyethylene glycol); etc.

[0381] In embodiments, a composition comprising at least one therapeutic RNA and at least one antagonist is formulated separately. Thus, the first component (as defined in the first aspect) and the second component (as defined in the first aspect) may be formulated (complexed / associated) as separate entities. The formulation / complex formation of the components may be the same (e.g., both components complexed in a polymer carrier) or different (e.g., one component encapsulated in an LNP and the other component complexed in polymer particles).

[0382] In embodiments, a composition comprising at least one therapeutic RNA and at least one antagonist is co-formulated. Thus, the first component (as defined in the first aspect) and the second component (as defined in the first aspect) are formulated (complexed / associated) as one entity. In these embodiments, the formulation / complexation of the components is the same (e.g., both components in an LNP).

[0383] In preferred embodiments, at least one therapeutic RNA and at least one antagonist are co-formulated to increase the probability that they are both present in one particle to ensure that at least one therapeutic RNA and at least one antagonist are taken up by the same cell.

[0384] In that context, a suitable cationic or polycationic compound for formulation can be selected from any one defined in the context of the first aspect. The first and second components of the composition may complex or associate with the same cationic or polycationic compound or with different cationic or polycationic compounds. In preferred embodiments, the first and second components of the composition may complex or associate within the same cationic or polycationic compound. In other embodiments, the first and second components of the composition may complex or associate within different cationic or polycationic compounds.

[0385] In a preferred embodiment of the composition, the polymeric carrier (of the first and / or second component) is a peptide polymer, preferably a polyethylene glycol / peptide polymer as defined above, and a lipid, preferably a lipidoid. In preferred embodiments, the first and second components of the composition may complex or associate (i.e., "co-formulate") within the same polymeric compound. In other embodiments, the first and second components of the composition may complex or associate (i.e., "formulated separately") within different polymeric compounds.

[0386] In a preferred embodiment of the composition, at least one therapeutic RNA of the first compound, preferably mRNA, is complexed, partially complexed, encapsulated, partially encapsulated, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes, and / or, at least one antagonist of the second compound, preferably a nucleic acid, is complexed, partially complexed, encapsulated, partially encapsulated, or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes.

[0387] Suitable liposomes / lipid nanoparticles can be derived from the disclosure provided in the context of the first aspect.

[0388] The first and second components of the composition can be complexed or associated within the same lipid nanoparticle or with different lipid nanoparticles. In a preferred embodiment, the first and second components of the composition can be complexed or associated (i.e., "co-formulated") within the same lipid nanoparticle. As described above, co-formulation increases the probability that both are present in one particle to ensure that at least one therapeutic RNA and at least one antagonist are taken up by the same cell.

[0389] In a preferred embodiment of the composition, at least one therapeutic RNA of the first compound is mRNA, and at least one antagonist of the second compound is an RNA oligonucleotide co-formulated in the liposomes / lipid nanoparticles as defined herein.

[0390] In an embodiment of the composition (or combination), the molar ratio of at least one antagonist of the second component as defined herein, preferably a nucleic acid, to at least one therapeutic RNA of the first component is in the range of about 1:1 to about 100:1, or in the range of about 20:1 to about 80:1.

[0391] In an embodiment of the composition (or combination), the molar ratio of at least one antagonist of the second component as defined herein, preferably a nucleic acid, to at least one therapeutic RNA of the first component is in the range of about 200:1 to about 1:1, or in the range of about 100:1 to about 1:1, or in the range of about 90:1 to about 1:1, or in the range of about 80:1 to about 1:1, or in the range of about 70:1 to about 1:1, or in the range of about 60:1 to about 1:1, or in the range of about 50:1 to about 1:1, or in the range of about 40:1 to about 1:1, or in the range of about 30:1 to about 1:1, or in the range of about 20:1 to about 1:1, or in the range of about 10:1 to about 1:1, or in the range of about 5:1 to about 1:1, or in the range of about 4:1 to about 1:1, or in the range of about 3:1 to about 1:1, or in the range of about 2:1 to about 1:1, or in the range of about 1:1 to about 1:200, or in the range of about 1:1 to about 1:100, or in the range of about 1:1 to about 1:90, or in the range of about 1:1 to about 1:80, or in the range of about 1:1 to about 1:70, or in the range of about 1:1 to about 1:60, or from about 1:1 to about 1:50, or from about 1:1 to about 1:40, or from about 1:1 to about 1:30, or from about 1:1 to about 1:20, or from about 1:1 to about 1:10, or from about 1:1 to about 1:5, or from about 1:1 to about 1:4, or from about 1:1 to about 1:3, or from about 1:1 to about 1:2.

[0392] In certain embodiments of the composition, the molar ratio of at least one antagonist of the second component, preferably a nucleic acid, to at least one therapeutic RNA of the first component, as defined herein, is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50; 1:59, 1:60, 1:70, 1:80, 1:90, 1:100.

[0393] In embodiments of the composition (or combination), the weight-to-weight ratio of at least one antagonist of the second component, preferably a nucleic acid, to at least one therapeutic RNA of the first component, as defined herein, is in the range of about 1:1 to about 1:30, or in the range of about 1:2 to about 1:20.

[0394] In preferred embodiments of the composition (or combination), the weight-to-weight ratio of at least one antagonist of the second component, preferably a nucleic acid, to at least one therapeutic RNA of the first component, as defined herein, is about 1:1 to about 1:20, or about 1:1 to about 1:15, or about 1:1 to about 1:10, or about 1:1 to about 1:9, or about 1:1 to about 1:8, or about 1:1 to about 1:7, or about 1:1 to about 1:6, or about 1:1 to about 1:5, or about 1:1 to about 1:4, or about 1:1 to about 1:3, or about 1:1 to about 1:2, or about 10:1 to about 1:1, or about 9:1 to about 1:1, or about 8:1 to about 1:1, or about 7:1 to about 1:1, or about 6:1 to about 1:1, or about 5:1 to about 1:1, or about 4:1 to about 1:1, or about 3:1 to about 1:1, or about 2:1 to about 1:1.

[0395] In certain embodiments of the composition, the weight-to-weight ratio of at least one antagonist of the second component, preferably a nucleic acid, as defined herein, to at least one therapeutic RNA of the first component is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, or 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1.

[0396] Particularly preferred is a weight-to-weight ratio of at least one antagonist of the second component, preferably a nucleic acid, as defined herein, to at least one therapeutic RNA of the first component in the range of about 1:2 to about 1:20, particularly about 1:5, 1:10, or 1:15.

[0397] Accordingly, the mass % (mass of total nucleic acids) of at least one antagonist of the second component, particularly a nucleic acid, in the composition or combination is about 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0398] In embodiments of the composition (or combination), the therapeutic RNA of the first compound is provided in an amount of about 20 ng to about 1000 μg, about 0.2 μg to about 900 μg, about 0.2 μg to about 800 μg, about 0.2 μg to about 700 μg, about 0.2 μg to about 600 μg, about 0.2 μg to about 500 μg, about 0.2 μg to about 400 μg, about 0.2 μg to about 300 μg, about 0.2 μg to about 100 μg, about 0.2 μg to about 100 μg, about 0.2 μg to about 80 μg, about 0.2 μg to about 60 μg, about 0.2 μg to about 40 μg, about 0.2 μg to about 20 μg, about 0.2 μg to about 10 μg, about 0.2 μg to about 5 μg, about 0.2 μg to about 2 μg, specifically, about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 1.2 μg, about 1.4 μg, about 1.6 μg, about 1.8 μg, about 1.8 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 16 μg, about 18 μg, about 20 μg, about 40 μg, about 60 μg, about 80 μg, about 100 μg.

[0399] In embodiments of the composition (or combination), the therapeutic RNA of the first compound is provided in an amount of about 20 μg to about 200 mg, about 0.2 mg to about 200 mg, about 0.2 mg to about 180 mg, about 0.2 mg to about 160 mg, about 0.2 mg to about 140 mg, about 0.2 mg to about 120 mg, about 0.2 mg to about 100 mg, about 0.2 mg to about 80 mg, about 0.2 mg to about 60 mg, about 0.2 mg to about 50 mg, about 0.2 mg to about 40 mg, about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.2 mg to about 10 mg, about 1 mg to about 10 mg, specifically, about 0.2 mg, about 0.4 mg, about 0.6 mg, about 0.8 mg, about 1 mg, about 1.2 mg, about 1.4 mg, about 1.6 mg, about 1.8 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 14 mg, about 16 mg, about 18 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg.

[0400] In embodiments of the composition (or combination), the antagonist of the second compound, preferably a nucleic acid, is provided in an amount of about 1 ng to about 50 μg, about 2 ng to about 100 μg, about 2 ng to about 80 μg, about 2 ng to about 60 μg, about 2 ng to about 40 μg, about 2 ng to about 20 μg, about 2 ng to about 10 μg, about 2 ng to about 5 μg, about 2 ng to about 2 μg, specifically, about 2 ng, about 4 ng, about 6 ng, about 8 ng, about 10 ng, about 12 ng, about 14 ng, about 16 ng, about 18 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 60 ng, about 70 ng, about 80 ng, about 90 ng, about 100 ng, about 110 ng, about 140 ng, about 160 ng, about 180 ng, about 200 ng, about 400 ng, about 600 ng, about 800 ng, about 1000 ng.

[0401] In embodiments of the composition (or combination), the antagonist of the second compound, preferably a nucleic acid, is provided in an amount of about 2 μg to about 20 mg, about 20 μg to about 20 mg, about 20 μg to about 18 mg, about 20 μg to about 16 mg, about 20 μg to about 14 mg, about 20 μg to about 12 mg, about 20 μg to about 10 mg, about 20 μg to about 8 mg, about 20 μg to about 6 mg, about 20 μg to about 4 mg, about 20 μg to about 2 mg, about 20 μg to about 1 mg, specifically, about 2 μg, about 4 μg, about 6 μg, about 8 μg, about 10 μg, about 12 μg, about 14 μg, about 16 μg, about 18 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 140 μg, about 160 μg, about 180 μg, about 200 μg, about 400 μg, about 600 μg, about 800 μg, about 1000 μg.

[0402] In a preferred embodiment, the composition comprises about 20 ng to about 100 μg of a therapeutic RNA, preferably mRNA, of the first compound as defined herein, and about 0.2 ng to about 10 μg of an antagonist, preferably a nucleic acid antagonist, of the second compound as defined herein.

[0403] In another preferred embodiment, the composition comprises from about 200 μg to about 200 mg of a therapeutic RNA, preferably mRNA, of a first compound as defined herein, and from about 20 μg to about 20 mg of an antagonist, preferably a nucleic acid antagonist, of a second compound as defined herein.

[0404] In a preferred embodiment, the composition comprising the first and second components is administered in a Ringer's or Ringer's lactate solution.

[0405] In a preferred embodiment, administration of the composition to a cell, tissue, or organism results in an increase or prolongation of the activity of the therapeutic RNA of the first component (contained in said composition) or at least equivalent activity as compared to administration of the corresponding first component alone.

[0406] The meaning of the term "activity" in this context depends on the mode of treatment of the therapeutic RNA of the first component. Thus, "activity" is closely related to the therapeutic effect of the therapeutic RNA of the first component. In embodiments where the therapeutic RNA is a coding RNA, "activity" must be understood as the expression that occurs after administration to a cell, tissue, or organism, such as protein expression, where the protein is provided by the cds of the administered coding RNA (e.g., mRNA). In embodiments where the therapeutic RNA is a coding RNA encoding an antigen, "activity" must be understood as the expression that occurs after administration to a cell, tissue, or organism, such as protein expression, where the protein is provided by the cds (e.g., mRNA) of the administered coding RNA and / or the induction of an antigen-specific immune response (e.g., B cell response and / or T cell response).

[0407] In particularly preferred embodiments, administration of the composition to a cell, tissue, or organism results in an increase or prolongation of the activity of the therapeutic RNA of the first component (contained in the composition) as compared to administration of the corresponding first component as a control.

[0408] In another particularly preferred embodiment, administration of the composition to a cell, tissue, or organism results in an increase or prolongation of the activity of the therapeutic RNA (comprising unmodified nucleotides) of the first component comprised in the composition, as compared to administration of a corresponding first component (wherein the RNA comprises modified nucleotides and has the same RNA sequence) as a control.

[0409] Thus, in a preferred embodiment of the composition, the activity of the therapeutic RNA (or corresponding control) is expression, preferably protein expression, preferably protein expression for the coding therapeutic RNA, such as therapeutic mRNA. Expression may be determined as defined in the context of the first aspect.

[0410] In a preferred embodiment, administration of the composition to a cell, tissue, or organism results in decreased (innate) immune activation as compared to administration of a therapeutic RNA or first component as a control.

[0411] In a further preferred embodiment, administration of the composition to a cell, tissue, or organism results in essentially the same or at least equivalent (innate) immune activation as compared to administration of a control RNA having the same RNA sequence and comprising modified nucleotides (such as defined herein).

[0412] Preferably, the decreased immune activation of the composition is a decreased level of at least one cytokine selected from Rantes, MIP-1α, MIP-1β, McP1, TNFα, IFNγ, IFNα, IFNβ, IL-12, IL-6, or IL-8. Cytokine levels can be determined as defined in the context of the first aspect.

[0413] In a preferred embodiment, administration of the composition is performed more than once, for example, once or more per day, once or more per week, once or more per month, or more than once per month. Advantageously, the compositions of the invention are suitable for repeated administration, for example, for chronic treatment.

[0414] The composition can be administered via oral, parenteral, inhaled spray, topical, rectal, nasal, oral, vaginal, or an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intramedullary, intraliver, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, intraocular, intravitreal, subretinal, intratumoral.

[0415] In particularly preferred embodiments, administration of the composition is by intravenous injection. In certain embodiments, the composition is administered intravenously as chronic therapy (e.g., once or more per day, such as once or more per day, once or more per week, once or more per month, or once or more per month).

[0416] In particularly preferred embodiments, the pharmaceutical composition (I) at least one first component, preferably at least one mRNA encoding a therapeutic peptide or protein, such as an antibody, enzyme, antigen, wherein preferably said mRNA optionally does not contain modified nucleotides, and wherein said mRNA contains a Cap1 structure (preferably obtainable by co-transcriptional capping), as the first component, and (II) at least one second component, preferably at least one single-stranded RNA oligonucleotide containing at least one 2'-O-methylated RNA nucleotide, preferably containing a nucleic acid sequence according to formula I, as the second component, wherein preferably said first and second components of the composition are co-formulated in lipid nanoparticles as defined herein or in polyethylene glycol / peptide polymers as defined herein.

[0417] [Kit or Kit of Parts] In a third aspect, the invention preferably provides a kit or kit of parts comprising the individual components of the combination (e.g., as defined in the context of the first aspect) and / or a pharmaceutical composition (e.g., as defined in the context of the second aspect).

[0418] In particular, embodiments related to the first and second aspects of the present invention are equally applicable to embodiments of the third aspect of the present invention, and embodiments related to the third aspect of the present invention are equally applicable to embodiments of the first and second aspects of the present invention.

[0419] In a preferred embodiment of the third aspect, the kit or kit of parts comprises at least one first and at least one second component as defined in the context of the first aspect, and / or at least one composition as defined in the context of the second aspect, optionally a liquid vehicle for solubilization, and optionally an instruction manual providing information regarding the administration and / or dosage of the components.

[0420] In a preferred embodiment, the kit or kit of parts comprises the following: (a) at least one first component as defined herein, preferably a therapeutic peptide or protein, such as an mRNA encoding an antibody, enzyme, antigen, wherein preferably the mRNA does not contain modified nucleotides, wherein preferably the mRNA contains a Cap1 structure, and wherein preferably the first component is formulated in lipid nanoparticles or in a polyethylene glycol / peptide polymer; (b) at least one second component as defined herein, preferably a single-stranded RNA oligonucleotide containing at least one 2'-O-methylated RNA nucleotide, preferably containing a nucleic acid sequence according to formula I, and preferably the second component is formulated in lipid nanoparticles or in a polyethylene glycol / peptide polymer; (c) optionally, a liquid vehicle for solubilizing (a) and / or (b), and optionally an instruction manual providing information regarding the administration and dosage of the components.

[0421] In a preferred embodiment, the kit or kit of parts comprises the following: (a) At least one composition defined in the context of the second aspect; (b) Optionally, a liquid vehicle for solubilization, and optionally, a technical manual providing information regarding the administration and dosage of the components.

[0422] The embodiments and features disclosed in the context of the first and second components, or the compositions of the second aspect, are similarly applicable to the RNA and / or compositions of a kit or a kit of parts.

[0423] The kit or kit of parts may further comprise components such as those described in the context of the first or second component, or a composition, in particular, a pharmaceutically acceptable carrier, excipient, buffer, etc.

[0424] The technical manual of the kit or kit of parts may include descriptions regarding administration, dosage, and patient groups. Such a kit, preferably a kit of parts, may be applied for any of the uses or medical uses described herein, for example.

[0425] Preferably, the individual components of the kit or kit of parts may be provided in lyophilized form. The kit may further contain, as part, a therapeutic RNA of the first component, and / or an antagonist of the second component, preferably a nucleic acid, and / or a vehicle (e.g., a pharmaceutically acceptable buffer solution) for solubilizing the composition of the second aspect.

[0426] In a preferred embodiment, the kit or kit of parts comprises a Ringer's solution or a Ringer's lactate solution.

[0427] In a preferred embodiment, the kit or kit of parts comprises a syringe needle, a microneedle, an injection device, a catheter, an implant delivery device, or a microcannula.

[0428] Any of the above kits can be used in a use or medical use as defined in the context of this specification.

[0429] 〔Medical Use〕 A further aspect relates to a first medical use of the provided combination, composition, or kit.

[0430] The embodiments described below (in the context of the "treatment method") are also applicable to the first medical use and further medical uses described herein.

[0431] Accordingly, the present invention provides a combination defined in the context of a first aspect for use as a medicament, a composition defined in a second aspect for use as a medicament, and a kit or kit of parts defined in a third aspect for use as a medicament.

[0432] In particular, the combination, composition, or kit or kit of parts may be used for human medical purposes and for veterinary medical purposes, preferably for human medical purposes.

[0433] In particular, the combination, composition, or kit or kit of parts is for use as a medicament for human medical purposes, where the combination, composition, or kit or kit of parts may be particularly suitable for infants, neonates, immunocompromised recipients, as well as pregnant and lactating women and the elderly.

[0434] A further aspect relates to a further medical use of the provided combination, composition, or kit.

[0435] Accordingly, the present invention provides a combination defined in the context of a first aspect for use as a medicament, a composition defined in a second aspect for use as a medicament, and a kit or kit of parts defined in a third aspect for use as a chronic treatment.

[0436] The term "chronic treatment" relates to a treatment that requires administration of a combination, composition, or kit or kit of parts one or more times, such as one or more times a day, one or more times a week, one or more times a month.

[0437] Furthermore, the present invention provides a combination defined in the context of the first aspect, a composition defined in the second aspect, and a kit or kit of parts defined in the third aspect for use in the treatment or prevention of an infection or a disorder associated with such an infection. Preferably, the infection is selected from viral infections, bacterial infections, protozoal infections. Thus, in said embodiment, the therapeutic RNA encodes at least one antigen.

[0438] The present invention further provides a combination defined in the context of the first aspect, a composition defined in the second aspect, and a kit or kit of parts defined in the third aspect for use in the treatment or prevention of a tumor disease or a disorder associated with such a tumor disease. Thus, in said embodiment, the therapeutic RNA may encode at least one tumor or cancer antigen and / or at least one therapeutic antibody (e.g., a checkpoint inhibitor).

[0439] The present invention further provides a combination as defined in the context of the first aspect, a composition as defined in the second aspect, and a kit or kit of parts as defined in the third aspect for use in the treatment or prevention of a genetic disorder or condition.

[0440] The present invention further provides a combination as defined in the context of the first aspect, a composition as defined in the second aspect, and a kit or kit of parts as defined in the third aspect for use in the treatment or prevention of protein or enzyme deficiencies or protein replacement. Thus, in said embodiment, the therapeutic RNA encodes at least one protein or enzyme. "Protein or enzyme deficiency" in this context must be understood as a disease or deficiency in which at least one protein is lacking, for example A1AT deficiency.

[0441] [[Methods of Treatment and Delivery]] A further aspect of the invention relates to a method of treating or preventing a disease, disorder, or condition.

[0442] (In the context of the first medical use and further medical uses) The above embodiments are also applicable to methods of treatment as described herein.

[0443] In a preferred embodiment of the third aspect, a method of treating or preventing a disorder, disease, or condition comprises the step of applying or administering to a subject the combination of the first aspect, the composition of the second aspect, or the kit or kit of parts of the second aspect.

[0444] The combination is preferably administered as a "co - administration". The term "co - administration" generally refers to the administration of at least two different substances in a sufficiently close period of time. Co - administration means the simultaneous administration, as well as the administration in a temporally spaced order up to several days, of at least two different substances either in a single dose or in separate doses, in any order.

[0445] In a preferred embodiment, the application or administration of the first and second components is carried out essentially simultaneously (as defined herein).

[0446] In some embodiments, the antagonists and therapeutic RNAs defined herein are administered simultaneously as part of the same composition. In some embodiments, the antagonists and therapeutic RNAs defined herein are administered simultaneously as different compositions. In some embodiments, the antagonist and the therapeutic RNA are administered by the same route of administration. In some embodiments, the antagonist and the therapeutic RNA are administered by different routes of administration.

[0447] In a preferred embodiment, the application or administration of the first component and the second component is carried out continuously (as defined herein). In some embodiments, the antagonist is administered before the therapeutic RNA. In some embodiments, the therapeutic RNA is administered before the antagonist. In some embodiments, the antagonist and the therapeutic RNA are administered by the same route of administration. In some embodiments, the antagonist and the therapeutic RNA are administered by different routes of administration.

[0448] In a preferred embodiment, the application or administration of the combination of the first aspect, the composition of the second aspect, or the kit of the third aspect or the kit of parts is carried out (as defined herein) one or more times a day, for example one or more times, one or more times a week, one or more times a month, or one or more times a month.

[0449] Administration can be by oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramedullary, intraliver, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, intraocular, intravitreal, subretinal, intratumoral.

[0450] In a preferred aspect, the step of applying or administering is subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramedullary, intraliver, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, intraocular, intravitreal, subretinal, or intratumoral.

[0451] In preferred embodiments, the subject in need is a mammalian subject such as a cow, pig, horse, sheep, cat, dog; and / or a bird (including commercially relevant birds such as poultry, chicken, duck, goose, and / or turkey). In particularly preferred embodiments, the subject in need is a human subject.

[0452] [Method for reducing or suppressing (innate) immune activation by therapeutic RNA] A further aspect of the invention relates to a method for reducing or suppressing (innate) immune activation induced by therapeutic RNA. By reducing or suppressing the immune activation induced by therapeutic RNA, the efficiency at the time of administration (e.g., translation of therapeutic RNA, activity of therapeutic RNA) can be increased. Thus, the "method for reducing or suppressing (innate) immune activation of therapeutic RNA" described herein should also be understood as a "method for increasing the effectiveness of therapeutic RNA".

[0453] In preferred embodiments, the method comprises administering to the subject at least one therapeutic RNA (as defined herein) and further at least one antagonist of at least one RNA-sensing pattern recognition receptor.

[0454] The at least one antagonist of at least one RNA-sensing pattern recognition receptor may be provided as a separate entity (as described, for example, in the context of the combinations of the first aspect), or may be provided as a single composition comprising at least one therapeutic RNA and further at least one antagonist of at least one RNA-sensing pattern recognition receptor.

[0455] Advantageously, administration of the antagonist reduces the innate immune response that can be induced by the therapeutic RNA (e.g., without affecting the translation of the therapeutic coding RNA, for example). Appropriately, reducing the stimulation of the innate immune response can be advantageous for various medical uses of the therapeutic RNA. In particular, the method can enable, for example, chronic administration of the therapeutic RNA, or can enhance or improve the therapeutic effect of the therapeutic RNA encoding an antigen (e.g., a viral antigen, a tumor antigen). Thus, reducing the innate immune response of the therapeutic RNA of the present invention leads to increased efficacy of the therapeutic RNA (e.g., upon administration to a cell or a subject).

[0456] Furthermore, in that context, the method enables reduction of the reactogenicity of the coding therapeutic RNA (e.g., including a cds encoding an antigen). Reactogenicity refers to the property of a vaccine, for example, that can cause side effects, particularly an excessive immune reaction and the associated signs and symptoms - fever, upper limb pain at the injection site, etc. Other symptoms of reactogenicity are typically bruising, redness, induration, and swelling.

[0457] Thus, a method of reducing or suppressing the (innate) immunostimulation of a therapeutic RNA is also understood as a method of reducing or suppressing the reactogenicity of the coding therapeutic RNA, wherein the coding RNA includes a cds encoding an antigen.

[0458] [[Method for increasing and / or prolonging the expression of (coding) therapeutic RNA]] A further aspect of the present invention relates to a method for increasing and / or prolonging the expression of a coding therapeutic RNA. By increasing and / or prolonging the expression of the coding therapeutic RNA, the efficiency upon administration (e.g., translation of the therapeutic RNA, activity of the therapeutic RNA) can be substantially increased. Thus, the "method for increasing and / or prolonging the expression of (coding) therapeutic RNA" described herein should also be understood as the "method for increasing the efficacy of (coding) therapeutic RNA".

[0459] In a preferred embodiment, the method comprises administering to a subject at least one RNA for coding therapy (as defined herein), and further at least one antagonist of at least one RNA-sensing pattern recognition receptor.

[0460] The at least one antagonist of the at least one RNA-sensing pattern recognition receptor may be provided as a separate entity (as described, for example, in the context of the combinations of the first aspect), or may be provided as a single composition comprising at least one therapeutic RNA and at least one antagonist of at least one RNA-sensing pattern recognition receptor.

[0461] Advantageously, the administration of the antagonist reduces the suppression of protein translation that can be induced by the therapeutic RNA. Suitably, the increase and / or prolongation may be advantageous for various medical uses of the therapeutic RNA. In particular, the method can enable, for example, chronic administration of the therapeutic RNA, or can enhance or improve, for example, the therapeutic effect of a therapeutic RNA encoding an antigen (such as a viral antigen, a tumor antigen). Thus, the increase and / or prolongation of the therapeutic RNA of the present invention leads to increased efficacy of the therapeutic RNA (such as upon administration to a cell or a subject).

[0462] 〔Summary of Lists and Tables〕 Table A: Preferred Small Molecule Antagonists of the Invention Table B: Preferred Oligonucleotide Antagonists of the Invention Table 1: Human Codon Usage Frequencies with Each Codon Frequency Shown for Each Amino Acid Table 2: Combinations of RNA Constructs of DOTAP Formulations Using 2'-O-Methylated Oligonucleotides Table 3: Constructs and Dosages of PpLuc mRNA and 2'-O-Methylated Oligonucleotides for Analysis of Expression and Immunostimulation In Vivo Table 4: Injection Schedule for Analysis of Expression and Immunostimulation In Vivo Table 5: Time points and experimental settings for the analysis of in vivo immune activation.

[0463] [Brief Description of the Drawings] Figure 1A shows the immunosuppressive effect by adding 2'-O-methylated oligonucleotide ("Gm18") to the immunostimulatory non-coding RNA ("RNAdjuvant") in PBMC in vitro. The DOTAP co-transfection of uncapped immunostimulatory non-coding RNA and 2'-O-methylated oligonucleotide shows a decrease in cytokine response compared to the transfection of immunostimulatory non-coding RNA measured only by the CBA array in the PBMC supernatant. Vehicle = DOTAP only; further details are provided in Example 2.

[0464] Figure 1B shows the immunosuppressive effect of in vitro addition of 2'-O-methylated oligonucleotide ("Gm18") to PpLuc mRNA in PBMC. The DOTAP co-transfection of capped coding PpLuc mRNA and oligonucleotide shows a decrease in cytokine response compared to the transfection of PpLuc mRNA only measured by the CBA array in the PBMC supernatant. Vehicle = DOTAP only; further details are provided in Example 2.

[0465] Figure 2 shows the expression of PpLuc from mRNA, 6 hours and 24 hours after intravenous injection of LNP in 129Sv mice, regardless of the presence or absence of the mixture of 2'-O-methylated RNA ("Gm18") oligonucleotide. To quantify PpLuc expression, bioluminescence was recorded for 3 minutes starting 5 minutes after intravenous injection of 3 mg of luciferin. The addition of 2'-O-methylated RNA oligonucleotide increases the expression of PpLuc at 24 hours after injection compared to PpLuc mRNA without 2'-O-methylated RNA oligonucleotide at either dose (10 μg or 30 μg of mRNA). Further details are provided in Example 2.

[0466] Figure 3 shows the expression of PpLuc in liver lysates after a single intravenous injection of PpLuc mRNA, with or without a mixture of 2'-O-methylated oligonucleotides ("Gm18") formulated in mouse LNPs. The livers were harvested 24 hours after injection of 10 μg or 30 μg of mRNA. Addition of the 2'-O-methylated RNA oligonucleotide increased PpLuc expression at 24 hours post-injection compared to PpLuc mRNA without the 2'-O-methylated RNA oligonucleotide at either dose. Further details are provided in Example 3.

[0467] Figure 4A shows the immunosuppressive effect of addition of the 2'-O-methylated oligonucleotide ("Gm18") to PpLuc mRNA 6 hours after injection, formulated in LNP in mice. A CBA array was performed using serum obtained 6 hours after intravenous injection to compare cytokine levels (Rantes, IL6, MCP1, MCP-1β, TNFα and IFNγ) induced by co-formulated mRNA + 2'-O-methylated oligonucleotide or by formulated mRNA alone. All cytokine levels strongly decreased by mixing the 2'-O-methylated oligonucleotide in a dose-dependent manner. Further details are provided in Example 3.

[0468] Figure 4B shows the immunosuppressive effect of addition of the 2'-O-methylated oligonucleotide ("Gm18") to PpLuc mRNA 24 hours after injection, formulated in LNP in mice. ELISA was performed using serum obtained 24 hours after intravenous injection to compare the levels of INFα induced by co-formulated mRNA + 2'-O-methylated oligonucleotide or by formulated mRNA alone. INFα levels strongly decreased by mixing the 2'-O-methylated oligonucleotide in a dose-dependent manner. Further details are provided in Example 3.

[0469] Figure 5A shows the immunosuppressive effect of in vitro addition of 2'-O-methylated oligonucleotide variants, RNA oligonucleotides, DNA oligonucleotides and small molecules to PpLuc mRNA. Co-transfection of capped coding PpLuc mRNA and oligonucleotides and small molecules with DOTAP shows a reduction in cytokine response (IFN-α) compared to transfection with PpLuc mRNA alone as measured by the CBA array in the PBMC supernatant. Vehicle = DOTAP only; further details are provided in Example 4.

[0470] Figure 5B shows the expression of PpLuc from mRNA with and without in vitro mixing of 2'-O-methylated oligonucleotide ("Gm18"), 2'-O-methylated oligonucleotide variants, RNA oligonucleotides, DNA oligonucleotides and small molecules to PpLuc mRNA. To quantify PpLuc expression, bioluminescence was recorded for 3 minutes starting 5 minutes after intravenous injection of 3 mg of luciferin. Addition of 2'-O-methylated oligonucleotide ("Gm18"), 2'-O-methylated oligonucleotide variants, RNA oligonucleotides, DNA oligonucleotides and small molecules increases the expression of PpLuc at 24 hours after transfection compared to PpLuc mRNA without the mixture.

[0471] 〔Example〕 The following examples are given to enable those skilled in the art to more clearly understand and practice the present invention. The present invention is not limited by the illustrated embodiments, which are intended only as examples of a single aspect of the present invention, and methods that are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the present invention will become readily apparent to those skilled in the art from the foregoing description, the accompanying drawings, and the following examples.

[0472] 〔Example 1: Generation of RNA constructs〕 〔1.1. Preparation of DNA templates〕 A DNA sequence encoding luciferase was prepared and used for subsequent in vitro transcription of RNA. The DNA sequence was prepared by modifying the wild-type cds sequence by introducing a GC-optimized cds. The sequence was introduced into a plasmid vector containing a UTR sequence, a stretch of adenosines, a histone-stem-loop structure, and optionally a stretch of 30 cytosines. The resulting plasmid DNA was transformed, grown in bacteria using a general protocol, the plasmid DNA was extracted, purified, and used for subsequent in vitro transcription of RNA as outlined below.

[0473] A DNA sequence encoding an immunostimulatory non-coding RNA was prepared and used for subsequent in vitro transcription of RNA. The resulting plasmid DNA was transformed, grown in bacteria using a general protocol, the plasmid DNA was extracted, purified, and used for subsequent in vitro transcription of RNA.

[0474] [1.2. In Vitro Transcription of RNA from Plasmid DNA Template] [1.2.1. Preparation of mRNA Encoding PPluc] The DNA plasmid prepared according to Section 1.1 was enzymatically linearized using a restriction enzyme and used for DNA-dependent in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP / GTP / CTP / UTP) and a cap analog (e.g., m7GpppG or m7G(5’)ppp(5’)(2’OMeA)pG or m7G(5’)ppp(5’)(2’OMeG)pG) in an appropriate buffer state. The resulting RNA was purified using RP-HPLC (PureMessenger®; WO2008 / 077592) and used for in vitro and in vivo experiments.

[0475] [1.2.2. Preparation of Immunostimulatory Non-Coding RNA] The DNA plasmid prepared according to Section 1.1 was enzymatically linearized using restriction enzymes and used for DNA-dependent RNA in vitro transcription with T7 RNA polymerase in the presence of a nucleotide mixture (ATP / GTP / CTP / UTP) in an appropriate buffer condition. The obtained non-coding RNA was purified using RP-HPLC (PureMessenger®; WO2008 / 077592) and used for in vitro and in vivo experiments.

[0476] [Example 2: Immunostimulation of human peripheral blood mononuclear cells (PBMC) by co-transfection of 2’-O-methylated oligonucleotide and RNA] For the examples described below, a 2’-O-methylated oligonucleotide (9-mer) was synthesized by Biomers (biomers.net GmbH, Germany): 5’-GAG CGmG CCA-3’ (SEQ ID NO: 85) (also referred to herein as “Gm18”).

[0477] [2.1 Preparation of human PBMC] Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy volunteers by standard Ficoll-Hypaque density gradient centrifugation (Ficoll 1.078 g / ml). PBMC were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS. After counting, the cells were resuspended at 50 million cells / ml in fetal bovine serum, 10% DMSO and frozen. Before use, the cells were thawed.

[0478] [2.2 PBMC stimulation] For transfection experiments, 2×10 per well 5Human PBMCs were seeded into each well of a 96-well plate in X-Vivo 15 medium (Lonza). For the preparation of the DOTAP complex containing both the immunostimulatory non-coding RNA and the 2'-O-methylated oligonucleotide (SEQ ID NO: 85), the oligonucleotide was first added to the immunostimulatory non-coding RNA at 25% by mass. For the preparation of the DOTAP complex containing both PpLuc mRNA and the 2'-O-methylated oligonucleotide, the oligonucleotide was first added to PpLuc mRNA at 25% by weight. Thus, the molar ratio of PpLuc mRNA to oligonucleotide was 1:45 (MW(oligonucleotide) = 2907 g / mol, MW(PpLuc mRNA) = 652377 g / mol). The DOTAP complex containing either the immunostimulatory non-coding RNA or PpLuc mRNA with or without the oligonucleotide was formed at a ratio of 1 μg of RNAdjuvant or 3 μl of DOTAP per 1 μg of mRNA. PBMCs were incubated overnight at 37 °C in a humidified 5% CO2 atmosphere in a total volume of 200 μl with 1 μg / ml of the immunostimulatory non-coding RNA or mRNA with or without 0.25 μg / ml of the oligonucleotide. To quantify the background stimulation, PBMCs were incubated with either DOTAP alone ("vehicle") or medium only. Twenty-four hours after transfection, the supernatant was collected.

[0479] [Table 4]

[0480] [2.3 Cytometric Bead Array (CBA)] In the supernatants collected from PBMCs stimulated with or without 2'-O-methylated oligonucleotides, the concentrations of IFN-α, IFN-γ, and TNF were measured by Cytometric Bead Array (CBA) using the following kits according to the manufacturer's instructions (BD Biosciences): Human Soluble Protein Master Buffer Kit (Catalog No. 558264), Assay Diluent (Catalog No. 560104), Human IFN-α Flex Set (Catalog No. 560379), Human IFN-γ Flex Set (Catalog No. 558269), Human TNF Flex Set (Catalog No. 560112); all kits were from BD Biosciences. Data were analyzed using FCAP Array v3.0 software (BD Biosciences).

[0481] [2.4 Results: Immunosuppressive effect by addition of 2'-O-methylated oligonucleotides] When 2'-O-methylated oligonucleotide ("Gm18") in human PBMCs is co-transfected with immunostimulatory non-coding RNA ("RNAdjuvant") using DOTAP, the immunosuppressive effect of 2'-O-methylated oligonucleotide is demonstrated by the decrease in the secretion of cytokines INF-α, INF-γ, and TNF as compared to the case of transfection with immunostimulatory non-coding RNA alone (Figure 1A).

[0482] When 2'-O-methylated oligonucleotide ("Gm18") is co-transfected with capped coding PpLuc mRNA into human PBMCs using DOTAP, the immunosuppressive effect of 2'-O-methylated oligonucleotide is demonstrated by the decrease in the secretion of cytokines INF-α, INF-γ, and TNF as compared to the case of transfection with PpLuc mRNA alone (Figure 1B).

[0483] The results indicate that the 2'-O-methylated oligonucleotides tested herein can reduce the immunostimulation of RNA, suggesting that combinations or compositions comprising an oligonucleotide and a therapeutic RNA can exhibit reduced immunostimulatory properties.

[0484] Example 3: Immunostimulation of PpLuc mRNA in combination with 2’O-methylated oligonucleotides and LNPs in vivo For the examples described below, a 9-mer 2'-O-methylated oligonucleotide (9-mer) was synthesized by Biomers (biomers.net GmbH, Germany): 5'-GAG CGmG CCA-3' (SEQ ID NO: 85).

[0485] 〔3.1 Preparation of PpLuc mRNA construct〕 An mRNA construct encoding PpLuc was prepared according to Example 1.

[0486] 〔3.2 LNP formulation〕 For the preparation of lipid nanoparticles (LNPs) containing both PpLuc mRNA and 2'-O-methylated oligonucleotides, first, the 2'-O-methylated oligonucleotide was added to PpLuc mRNA at either 20% or 6.7% mass percentage (see Table 3). Using cationic lipid, cholesterol, PEG-lipid and neutral lipid, LNPs containing PpLuc mRNA were prepared with or without the 2'-O-methylated oligonucleotide. The mRNA was diluted to 1 g / L in citrate buffer, pH 4. The ethanolic lipid solution was mixed with the aqueous RNA solution at a ratio of 1:3 (vol / vol) using a Nanoassemblr (Precision NanoSystems). Ethanol was then removed and the buffer was replaced by dialysis with 10 mM HEPES (pH 7.4) containing 9% sucrose. Finally, the LNP-formulated RNA was adjusted to 0.2 g / L.

[0487]

Table 5

[0488]

Table 6

[0489] 〔3.3 Intravenous injection of PpLuc mRNA, 2'-O-methylated oligonucleotide and LNP in mice〕 For in vivo experiments, various LNP formulations were injected into 8-week-old female mice (about 25 g, 129SV strain) (see Tables 4 and 5). Four animals were used per group. 10 μg or 30 μg of mRNA formulated with or without 2'-O-methylated oligonucleotide was intravenously injected at a concentration of 0.2 g / l. Bioluminescence imaging was performed 6 hours and 24 hours after LNP injection. Blood was collected 6 hours after LNP injection and finally 24 hours after LNP injection. Immediately thereafter, the mice were sacrificed, the livers were collected, placed in 1.5 ml PP tubes, frozen and stored until analysis (<-70 °C).

[0490] 〔3.4 Expression analysis from in vivo imaging〕 The expression of PpLuc was visualized 6 hours and 24 hours after a single intravenous injection of LNP-formulated PpLuc mRNA with or without a mixture of 2'-O-methylated oligonucleotide ("Gm18"). PpLuc expression was quantified from bioluminescence images recorded for 3 minutes starting 5 minutes after intravenous injection of 3 mg of luciferin (see Table 5). The addition of 2'-O-methylated oligonucleotide ("Gm18") increased the expression of PpLuc at 24 hours after injection compared to PpLuc mRNA without Gm18 at any dose (10 μg or 30 μg of mRNA) (see Figure 2).

[0491]

Table 7

[0492] 〔3.5 Expression analysis from cell lysates〕 To prepare the tissue lysates, steel beads were first added to each liver. The frozen liver was placed on a tissue lyser and shaken for 3 minutes. Then, 800 μl of lysis buffer was added (25 mM Tris-HCl pH 7.5, 2 mM EDTA, 10% (w / v) glycerol, 1% (w / v) Triton X-100, 2 mM DTT, and 1 mM PMSF). Tissue lysis was continued for an additional 6 minutes. The samples were centrifuged at 13,500 rpm for 10 minutes at 4 °C. 20 μl of each supernatant was added to a white LIA assay plate. The plate was introduced into a plate reader (Berthold Technologies TriStar2 LB 942), and 50 μl / well of beetle juice (PJK GmbH) containing luciferin as a substrate for firefly luciferase was injected. Luciferase activity was quantified as relative light units (RLU). The addition of 2'-O-methylated oligonucleotides increased the expression of PpLuc in the lysates 24 hours after injection compared to PpLuc mRNA without oligonucleotides at either dose (10 μg or 30 μg) (see Figure 3).

[0493] To analyze the effect of 2'-O-methylated oligonucleotides on immune activation, as described in Paragraph 2.3, the concentrations of IFNγ, TNFα, IL-6, MIP-1β, Rantes, and MCP1 in serum collected from blood taken 6 hours after LNP injection were measured by cytometric bead array (CBA). The addition of 2'-O-methylated RNA oligonucleotides to PpLuc mRNA strongly decreased the release of all inflammatory cytokines in a dose-dependent manner (see Figure 4A). To further evaluate the effect of 2'-O-methylated oligonucleotides on immune activation, the concentration of IFNα in serum from blood taken 24 hours after LNP injection was measured by ELISA. The addition of 2'-O-methylated oligonucleotides to PpLuc mRNA strongly decreased the release of IFNα in a dose-dependent manner (see Figure 4B).

[0494] ​ 〔Summary of Results (Examples 1 - 3)〕 The results of the in vitro experiment described in Example 2 and FIG. 1 show that the 2'-O-methylated oligonucleotide ("Gm18") used herein antagonizes the immune activation of co-administered RNA, typically induced by RNA-sensing pattern recognition receptors. Thus, the oligonucleotide acts as an antagonist of RNA-sensing pattern recognition receptors. The results indicate that combinations or compositions comprising an oligonucleotide antagonist and a therapeutic RNA advantageously reduce the immune activation properties of the therapeutic RNA. The results of the in vivo experiments described in Example 3 and FIGS. 2 - 4 show that the 2'-O-methylated oligonucleotide used herein also antagonizes the immune activation of RNA in vivo. Unexpectedly, the addition of the 2'-O-methylated oligonucleotide also increases / extends the expression of the RNA-encoded protein, suggesting that combinations or compositions comprising an oligonucleotide antagonist and a therapeutic RNA show an increase in expression and / or activity in vivo in addition to a decrease in immune activation, which is most important for most RNA-based drugs.

[0495] 〔Example 4. Immune Activation and Expression Efficiency of Human Peripheral Blood Mononuclear Cells (PBMCs) by Co-Transfection of RNA and 2'-O-Methylated Oligonucleotide Variants, RNA Oligonucleotides, DNA Oligonucleotides, and Small Molecules〕 For the examples described below, various oligonucleotides and small molecules were synthesized by Biomers (biomers.net GmbH, Germany), Invivogen (https: / / www.invivogen.com / (Link), United States) or Miltenyi Biotec (miltenyibiotec.com / DE-en / , Germany) (Table 6).

[0496] 〔4.1 Preparation of PpLuc mRNA Construct〕 An mRNA construct encoding PpLuc was prepared according to Example 1.

[0497]

Table 8

[0498] [Analysis of Expression and Immunoactivation of PMBC Co-Transfected with [4.2 2'-O-Methylated Oligonucleotide Variants, RNA- and DNA-Oligonucleotides and Small Molecules]] Human PBMCs were prepared according to Example 2.1. For the transfection experiment, 2 × 10 per well 5Individual human PBMCs were seeded into each well of a 96-well plate in X-Vivo 15 medium (Lonza). For the preparation of DOTAP (vehicle) complexes containing both an antagonist (either a 2'-O-methylated oligonucleotide variant, RNA oligonucleotide, DNA oligonucleotide or small molecule) and PpLuc mRNA (the same RNA design as shown in SEQ ID NO: 82, Table 2), the antagonist was first added to PpLuc mRNA at 20% mass percent (1:5 mRNA:oligo / small molecule). Thus, the molar ratio of PpLuc mRNA to antagonist was 1:45 (MW(oligonucleotide)=2907 g / mol, MW(PpLuc mRNA)=652377 g / mol). DOTAP complexes containing PpLuc mRNA and the antagonist were formed at a ratio of 5 μl of DOTAP per 1 μg of mRNA and 100 ng was transfected. PBMCs were incubated overnight with the mRNA at a total volume of 200 μl in a humidified 5% CO2 atmosphere at 37 °C with or without 0.25 μg / ml of the antagonist. To quantify background stimulation, PBMCs were incubated with DOTAP alone ("vehicle") or RPMI ("medium") only. Twenty-four hours after transfection, the supernatant was collected, the cells were lysed and stored at -80 °C. A Cytometric Bead Array (CBA) was performed according to 2.3. Expression analysis was performed by measuring luciferase acidity, measured as relative light units (RLU) with a BioTek SynergyHT plate reader. PpLuc activity was measured using 50 μl of lysate and 200 μl of luciferin buffer (75 μM luciferin, 25 mM glycylglycine, pH 7.8 (NaOH), 15 mM MgSO4, 2 mM ATP) with a measurement time of 5 seconds.

[0499] [4.3 Analysis of the immunosuppressive effect and expression efficiency of PMBCs co-transfected with 2'-O-methylated oligonucleotide variants, RNA- and DNA oligonucleotides and small molecules] When 2'-O-methylated oligonucleotide, RNA oligonucleotide, DNA oligonucleotide, and variants of small molecules are co-transfected into human PBMCs with capped coding PpLuc mRNA using DOTAP, an immunosuppressive effect is demonstrated by a decrease in the secretion of cytokine IFN-α, as measured by CBA array in the PBMC supernatant, compared to the case of transfection with only PpLuc mRNA (Figure 4A).

[0500] The addition of 2'-O-methylated oligonucleotide variants, RNA- and DNA oligonucleotides, and small molecules increases the expression of PpLuc in PBMCs 24 hours after transfection compared to PpLuc mRNA itself (Figure 4B).

[0501] [Others] [Form 1] A combination comprising (i) at least one first component comprising at least one therapeutic RNA, and (ii) at least one second component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor. A combination comprising or consisting of these. [Form 2] The combination according to Form 1, wherein the at least one RNA-sensing pattern recognition receptor induces cytokines upon binding of an RNA agonist. [Form 3] The combination according to Form 1 or 2, wherein the at least one RNA-sensing pattern recognition receptor inhibits translation upon binding of an RNA agonist. [Form 4] The combination according to any one of Forms 1 to 3, wherein at least one antagonist of the second component reduces cytokine induction by the at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist and / or reduces translation inhibition by the at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist. [Form 5] Administration of a combination of at least one therapeutic RNA of the first component and at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component results in a reduction of the innate immune response compared to administering at least one therapeutic RNA of the first component without combining it with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component, the combination according to any one of Forms 1 to 4. [Form 6] The induction of the innate immune response is determined by measuring the induction of cytokines, the combination according to Form 5. [Form 7] The cytokine is selected from the group consisting of IFN-α, TNF-α, IP-10, IFN-γ, IL-6, IL-12, IL-8, Rantes, MIP-1α, MIP-1β, McP1, or IFNβ, the combination according to Form 6. [Form 8] The induction of cytokines is measured by administering the combination to cells, tissues or organisms, preferably hPBMC, Hela cells or HEK cells, the combination according to Form 6 or 7. [Form 9] The at least one RNA-sensing pattern recognition receptor is an endosomal receptor or a cytoplasmic receptor, preferably an endosomal receptor, the combination according to any one of Forms 1 to 8. [Form 10] The at least one RNA-sensing pattern recognition receptor is a receptor for single-stranded RNA (ssRNA) and / or a receptor for double-stranded RNA (dsRNA), the combination according to any one of Forms 1 to 9. [Form 11] The at least one RNA-sensing pattern recognition receptor is selected from Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs), NOD-like receptors, PKR, OAS, SAMHD1, ADAR1, IFIT1 and / or IFIT5, the combination according to any one of Forms 1 to 10. [Form 12] The combination according to Form 11, wherein the at least one Toll-like receptor is selected from TLR3, TLR7, TLR8, and / or TLR9. [Form 13] The combination according to Form 11 or 12, wherein the at least one Toll-like receptor is selected from TLR8 and / or TLR9, most preferably selected from TLR7 and / or TLR8. [Form 14] The combination according to Form 11, wherein the retinoic acid-inducible gene-I-like receptor (RLR) is selected from RIG-1, MDA5, LGP2, cGAS, AIM2, NLRP3, NOD2, preferably RIG1 and / or MDA5. [Form 15] The combination according to any one of Forms 1 to 14, wherein at least one antagonist of the second component is selected from nucleotides, nucleotide analogs, nucleic acids, peptides, proteins, small molecules, lipids, or fragments, mutants, or derivatives thereof. [Form 16] The combination according to any one of Forms 1 to 15, wherein at least one antagonist of the second component is a nucleic acid. [Form 17] The combination according to any one of Forms 1 to 16, wherein at least one antagonist of the second component is a single-stranded nucleic acid. [Form 18] The combination according to Form 16 or 17, wherein the nucleic acid of the second component comprises nucleotides selected from DNA nucleotides, RNA nucleotides, PNA nucleotides, and / or LNA nucleotides, or analogs or derivatives thereof, or consists of these. [Form 19] The combination according to any one of Forms 16 to 18, wherein the nucleic acid of the second component comprises at least one modified nucleotide and / or at least one nucleotide analog or nucleotide derivative. [Form 20] The combination according to Form 19, wherein the at least one modified nucleotide and / or at least one nucleotide analog is selected from a backbone-modified nucleotide, a sugar-modified nucleotide and / or a base-modified nucleotide, or any combination thereof. [Form 21] The combination according to Form 19 or 20, wherein the at least one modified nucleotide and / or at least one nucleotide analog is selected from 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2'-O-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysidine, 1-methylguanosine, 7-methylguanosine, 2'-O-methylguanosine, queosine, epoxyqueosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, dihydrouridine, 5-methyluridine, 2'-O-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, methyl ester of uridine 5-oxyacetic acid, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, or 5-(isopentenylaminomethyl)2'-O-methyluridine. [Form 22] The combination according to any one of Forms 19 to 21, wherein at least one modified nucleotide is a sugar-modified nucleotide, preferably a 2'-ribose-modified RNA nucleotide. [Form 23] The combination according to Form 22, wherein the 2'-ribose-modified RNA nucleotide is a 2'-O-methylated RNA nucleotide. [Form 24] The combination according to Form 23, wherein the 2'-O-methylated RNA nucleotide is selected from 2'-O-methylated guanosine (Gm), 2'-O-methylated uracil (Um), 2'-O-methylated adenosine (Am), 2'-O-methylated cytosine (Cm), or 2'-O-methylated analogs of any of these nucleotides. [Form 25] The nucleic acid of the second component comprises at least one trinucleotide M-X-Y motif, wherein M is selected from Gm, Um, or Am, preferably M is Gm, wherein X is selected from G, A, or U, preferably X is G, wherein Y is selected from G, A, U, C, or dihydrouridine, preferably Y is C, the combination according to any one of Forms 16 to 24. [Form 26] The nucleic acid of the second component has the formula I N W -M-X-Y-N Z (Formula I) and comprises or consists of a nucleic acid sequence according to wherein N is independently selected from G, A, U, C, Gm, Am, Um, Cm, or a modified nucleotide, wherein W is 0 or an integer from 1 to 15, wherein Z is 0 or an integer from 1 to 15, wherein M, X, and Y are selected as defined in Form 25, the combination according to any one of Forms 16 to 25. [Form 27] The nucleic acid of the second component comprises, or consists of, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I, and each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I is identical to each other or is independently selected, the combination according to any one of forms 16 to 26. [Form 28] The nucleic acid of the second component comprises a 5' end lacking a triphosphate group, the combination according to any one of forms 16 to 27. [Form 29] The nucleic acid of the second component comprises a triphosphate group at the 5' end, the combination according to any one of forms 16 to 27. [Form 30] The nucleic acid of the second component has a length of about 3 to about 50 nucleotides, about 5 to about 25 nucleotides, about 5 to about 15, or about 5 to about 10 nucleotides, preferably about 5 to about 15 nucleotides, the combination according to any one of forms 16 to 29. [Form 31] The nucleic acid of the second component has a length of 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, or 13 nucleotides, preferably 9 nucleotides, the combination according to any one of forms 16 to 30. [Form 32] The nucleic acid of the second component is a single-stranded oligonucleotide, the combination according to any one of forms 16 to 31. [Form 33] The single-stranded oligonucleotide is a single-stranded RNA oligonucleotide, the combination according to form 32. [Form 34] The nucleic acid of the second component comprises, or consists of, a nucleic acid sequence derived from bacterial tRNA, preferably bacterial tRNA Tyr the combination according to any one of forms 16 to 33. [Form 35] The nucleic acid sequence is bacterial tRNA Tyr preferably bacterial tRNA TyrThe D-loop, most preferably the E. coli tRNA Tyr The D-loop of Tyr or a combination according to Form 34 derived therefrom. [Form 36] The nucleic acid of the second component is identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85 to 212, or a fragment of any of these sequences, or contains a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of these nucleic acid sequences, the combination according to any one of Forms 16 to 35. [Form 37] The nucleic acid of the second component is identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85 to 87, 149 to 212, or a fragment of any of these sequences, or contains a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of these nucleic acid sequences, Preferably, The nucleic acid of the second component is identical to a nucleic acid sequence according to 5'-GAG CGmG CCA-3' (SEQ ID NO: 85), or a fragment thereof, or contains a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of that nucleic acid sequence, the combination according to Form 36. [Form 38] At least one therapeutic RNA of the first component is selected from coding RNA, non-coding RNA, circular RNA (circRNA), RNA oligonucleotide, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, mRNA, riboswitch, ribozyme, RNA aptamer, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA, small nuclear RNA (snRNA), self-replicating RNA, replicon RNA, small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA), and is the combination according to any one of Forms 1 to 37. [Form 39] At least one therapeutic RNA of the first component is RNA transcribed in vitro, and is the combination according to any one of Forms 1 to 38. [Form 40] The RNA transcribed in vitro can be obtained by in vitro transcription of RNA using a sequence-optimized nucleotide mixture, and is the combination according to Form 39. [Form 41] At least one therapeutic RNA of the first component is purified RNA, and is the combination according to any one of Forms 1 to 40. [Form 42] The purified RNA is purified by RP-HPLC and / or TFF and / or oligo d(T) purification, and is the combination according to Form 41. [Form 43] At least one therapeutic RNA of the first component is coding RNA, and is the combination according to any one of Forms 1 to 42. [Form 44] The coding RNA is selected from mRNA, self-replicating RNA, circular RNA, viral RNA, or replicon RNA, and is the combination according to Form 43. [Form 45] The combination according to any one of Forms 1 to 44, wherein at least one therapeutic RNA of the first component is mRNA. [Form 46] The combination according to any one of Forms 43 to 45, wherein the coding RNA or mRNA comprises at least one coding sequence encoding at least one peptide or protein. [Form 47] The combination according to Form 46, wherein the expression of at least one peptide or protein encoded by the coding RNA or mRNA is increased or extended upon administration to a cell, tissue or organism, as compared to expressing at least one peptide or protein encoded by the coding RNA or mRNA without combining with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component, by combining with at least one antagonist of at least one RNA-sensing receptor of the second component. [Form 48] The combination according to Form 46 or 47, wherein the at least one peptide or protein is a therapeutic peptide or therapeutic protein or is derived therefrom. [Form 49] The combination according to Form 48, wherein the therapeutic peptide or therapeutic protein is an antibody, cytokine, receptor, receptor agonist, receptor antagonist, binding protein, CRISPR-related endonuclease, chaperone, transporter protein, ion channel, membrane protein, secreted protein, transcription factor, enzyme, peptide hormone or protein hormone, growth factor, structural protein, cytoplasmic protein, cytoskeletal protein, viral antigen, bacterial antigen, protozoan antigen, allergen, tumor antigen, or a fragment, or variant, combination thereof, or is derived therefrom. [Form 50] The at least one coding sequence is a codon-optimized coding sequence, The combination according to any one of Forms 46 to 49, wherein the amino acid sequence encoded by the at least one codon-modified coding sequence is preferably not modified as compared with the amino acid sequence encoded by the corresponding wild-type coding sequence. [Form 51] The combination according to Form 50, wherein the at least one codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage frequency-conforming coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof. [Form 52] The combination according to any one of Forms 1 to 51, wherein at least one therapeutic RNA of the first component, preferably mRNA, contains a 5'-cap structure. [Form 53] The combination according to Form 52, wherein the 5'-cap structure is a cap0, cap1, cap2, modified cap0 or modified cap1 structure. [Form 54] The combination according to Form 53, wherein the 5'-cap structure is a cap1 structure. [Form 55] The combination according to Form 54, wherein the cap1 structure can be obtained by co-transcriptional capping using a trinucleotide cap1 analog. [Form 56] The combination according to any one of Forms 1 to 55, wherein about 70%, 75%, 80%, 85%, 90%, 95% of the therapeutic RNA(s) of the first component contains a capping 1 structure as determined using a capping detection assay. [Form 57] The combination according to any one of Forms 1 to 56, wherein at least one therapeutic RNA of the first component contains at least one modified nucleotide or modified nucleotide analog. [Form 58] The combination according to form 57, wherein the at least one modified nucleotide is selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine. [Form 59] The combination according to any one of forms 1 to 58, wherein at least one therapeutic RNA of the first component, preferably mRNA, comprises at least one poly(A) sequence, and / or at least one poly(C) sequence, and / or at least one histone stem-loop sequence / structure. [Form 60] The poly(A) sequence is located at the 3' end of the therapeutic RNA. And / or The 3' end of the RNA consists of a poly(A) sequence terminated with A nucleotides. The combination according to form 59. [Form 61] The combination according to any one of forms 1 to 60, wherein at least one therapeutic RNA of the first component, preferably mRNA, comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. [Form 62] The at least one heterologous 3'-UTR is a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or The combination according to form 61, comprising a nucleic acid sequence derived from a homolog, fragment or variant of any of these genes. [Form 63] The at least one heterologous 5'-UTR is a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUB4B and UBQLN2, or The combination according to form 61, comprising a nucleic acid sequence derived from a homolog, fragment or variant of any of these genes. [Form 64] At least one antagonist of said second component, preferably a nucleic acid, and / or, At least one therapeutic RNA of said first component, One or more cationic or polycationic compounds, preferably a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, or a cationic or polycationic peptide, or any combination thereof, Complexes or associates with, or at least partially complexes or partially associates with, the combination according to any one of Forms 1 to 63. [Form 65] The combination according to Form 64, wherein said one or more cationic or polycationic peptides are selected from SEQ ID NOs: 39 to 43, or any combination thereof. [Form 66] The cationic or polycationic polymer is, A polyethylene glycol / peptide polymer containing HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (SEQ ID NO: 42 of the peptide monomer), and / or, The combination according to Form 64, which is a polyethylene glycol / peptide polymer containing HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-OH (SEQ ID NO: 43 of the peptide monomer). [Form 67] The combination according to Form 65 or 66, further comprising a lipid and / or lipidoid. [Form 68] At least one antagonist of said second component, preferably a nucleic acid, and / or, At least one therapeutic RNA of said first component, Complexed with, partially complexed with, encapsulated by, partially encapsulated by, or associated with one or more lipids, Thereby forming a combination according to any one of Forms 1 to 67 that forms liposomes, lipid nanoparticles (LNP), lipoplexes, and / or nanoliposomes, preferably lipid nanoparticles (LNP). [Form 69] Said LNP being (i) at least one cationic lipid, preferably lipid III-3, (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), (iii) at least one steroid or steroid analog, preferably cholesterol, and, (iv) at least one PEG lipid, preferably a PEGylated lipid of formula (IVa) comprising Preferably, (i) to (iv) are in a molar ratio of about 20 to 60% cationic lipid; 5 to 25% neutral lipid; 25 to 55% sterol; 0.5 to 15% PEG lipid, the combination according to Form 68. [Form 70] A pharmaceutical composition comprising or consisting of a combination according to any one of Forms 1 to 69 and optionally at least one pharmaceutically acceptable carrier. [Form 71] The pharmaceutical composition according to Form 70, wherein said at least one therapeutic RNA and said at least one antagonist are formulated separately. [Form 72] The pharmaceutical composition according to Form 71, wherein at least one therapeutic RNA and at least one antagonist are co-formulated to increase the probability that both are present in one particle and ensure that at least one therapeutic RNA and at least one antagonist are taken up by the same cell. [Form 73] The pharmaceutical composition according to any one of Forms 70 to 72, wherein the molar ratio of at least one antagonist, preferably a nucleic acid, to at least one therapeutic RNA is in the range of about 1:1 to about 100:1, or in the range of about 20:1 to about 80:1. [Form 74] The pharmaceutical composition according to any one of Forms 70 to 73, wherein the weight-to-weight ratio of at least one antagonist, preferably a nucleic acid, to at least one therapeutic RNA is in the range of about 1:1 to about 1:30, or in the range of about 1:2 to about 1:10. [Form 75] The pharmaceutical composition according to any one of Forms 70 to 74, wherein administration of the composition to a cell, tissue, or organism results in essentially the same or at least equivalent activity of the therapeutic RNA as compared to administration of the corresponding therapeutic RNA alone. [Form 76] The pharmaceutical composition according to any one of Forms 70 to 74, wherein administration of the composition to a cell, tissue, or organism results in an increase in the activity of the therapeutic RNA as compared to, for example, administration of the corresponding therapeutic RNA alone. [Form 77] The pharmaceutical composition according to Form 75 or 76, wherein the activity of the therapeutic RNA is the expression of the encoded peptide or protein, preferably the expression of the protein. [Form 78] The pharmaceutical composition according to any one of Forms 70 to 77, wherein administration of the composition to a cell, tissue, or organism results in a reduction in (innate) immune activation as compared to administration of the corresponding therapeutic RNA alone. [Form 79] At least one first component and at least one second component according to any one of Forms 1 to 69, and / or At least one pharmaceutical composition according to any one of Forms 70 to 78, Optionally, A liquid vehicle for solubilization, and, Optionally, a technical manual providing information on the administration and / or dosage of the components A kit or a kit of parts comprising the same. [Form 80] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use as a medicament. [Form 81] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use in chronic treatment. [Form 82] The medical use according to Form 81, wherein in chronic treatment, the administration of the combination, the pharmaceutical composition, the kit or the kit of parts is carried out one or more times, for example, one or more times a day, one or more times a week, or one or more times a month. [Form 83] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use in the treatment or prevention of an infectious disease, preferably a viral infectious disease, a bacterial infectious disease, or a protozoan infectious disease. [Form 84] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use in the treatment or prevention of a tumor disease or a disorder associated with such a tumor disease. [Form 85] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use in the treatment or prevention of a genetic disorder or condition. [Form 86] The combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79 for use in the treatment or prevention of a protein or enzyme deficiency. [Form 87] A method for treating or preventing a disorder, disease, or condition, the method comprising administering or applying to a subject in need thereof the combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79. [Form 88] The method according to Form 87, wherein the administration of the first component and the second component is essentially simultaneous. [Form 89] The method according to Form 87, wherein the administration of the first component and the second component is continuous. [Form 90] The method according to any one of Forms 86 to 89, wherein the administration of the combination, the pharmaceutical composition, the kit or the kit of parts is carried out one or more times, for example one or more times a day, one or more times a week, one or more times a month. [Form 91] The method according to any one of Forms 86 to 90, wherein the administration or application is subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intranasal, intra-oral, intra-sternal, intramedullary, intra-hepatic, intralesional, intracranial, transdermal, intradermal, intra-pulmonary, intraperitoneal, intra-cardiac, intra-arterial, intra-ocular, intravitreal, subretinal, or intratumoral. [Form 92] The method according to any one of Forms 86 to 91, wherein the subject in need thereof is a mammalian subject, preferably a human subject. [Form 93] A method for reducing the (innate) immune activation of a therapeutic RNA, the method comprising administering or applying to a subject in need thereof the combination according to any one of Forms 1 to 69, the pharmaceutical composition according to any one of Forms 70 to 78, or the kit or kit of parts according to Form 79. [Form 94] A method for increasing and / or prolonging the expression of a peptide or protein encoded by a therapeutic RNA to be encoded, the method comprising applying or administering to a subject in need thereof a combination according to any one of Forms 1 to 69, a pharmaceutical composition according to any one of Forms 70 to 78, or a kit or kit of parts according to Form 79.

Brief Description of the Drawings

[0502]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 5A

Claims

**Claim 1** A combination, wherein the combination (i) at least one first component comprising at least one therapeutic RNA, and (ii) at least one second component comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor, is a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome, preferably a lipid nanoparticle (LNP), wherein at least one therapeutic RNA of the first component is selected from coding RNAs, and at least one antagonist of the second component, preferably a nucleic acid, and at least one therapeutic RNA of the first component are complexed, partially complexed, encapsulated, partially encapsulated, or associated with one or more lipids. A combination. **Claim 2** wherein the at least one RNA-sensing pattern recognition receptor induces cytokines upon binding of an RNA agonist, and / or wherein the at least one RNA-sensing pattern recognition receptor inhibits translation upon binding of an RNA agonist. The combination according to claim 1. **Claim 3** wherein at least one antagonist of the second component reduces cytokine induction by the at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist, and / or reduces translation inhibition by the at least one RNA-sensing pattern recognition receptor upon binding of an RNA agonist, and / or administration of a combination of at least one therapeutic RNA of the first component and at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component results in a reduction of the innate immune response compared to administration of at least one therapeutic RNA of the first component without combining it with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component. The combination according to claim 1 or 2. **Claim 4** wherein the at least one RNA-sensing pattern recognition receptor is a receptor for single-stranded RNA (ssRNA) and / or a receptor for double-stranded RNA (dsRNA). The combination according to any one of claims 1 to 3. **Claim 5** At least one RNA-sensing pattern recognition receptor is selected from Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs), NOD-like receptors, PKR, OAS, SAMHD1, ADAR1, IFIT1, and / or IFIT5, preferably, said at least one Toll-like receptor is selected from TLR3, TLR7, TLR8, and / or TLR9, more preferably, at least one Toll-like receptor is selected from TLR8 and / or TLR9, most preferably selected from TLR7 and / or TLR8, and optionally, said retinoic acid-inducible gene-I-like receptor (RLR) is selected from RIG-1, MDA5, LGP2, cGAS, AIM2, NLRP3, NOD2, preferably RIG1 and / or MDA5, the combination according to any one of claims 1 to 4.

6. At least one antagonist of the second component is a nucleic acid, preferably a single-stranded nucleic acid, and / or, the nucleic acid of the second component comprises nucleotides selected from DNA nucleotides, RNA nucleotides, PNA nucleotides, and / or LNA nucleotides, or analogs or derivatives of any of these, or consists of these, the combination according to any one of claims 1 to 5.

7. The nucleic acid of the second component comprises at least one modified nucleotide and / or at least one nucleotide analog or nucleotide derivative, said at least one modified nucleotide and / or at least one nucleotide analog is preferably selected from backbone-modified nucleotides, sugar-modified nucleotides, and / or base-modified nucleotides, or any combination thereof, More preferably, at least one modified nucleotide and / or at least one nucleotide analog is selected from 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2'-O-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysidine, 1-methylguanosine, 7-methylguanosine, 2'-O-methylguanosine, queuosine, epoxyqueuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, dihydrouridine, 5-methyluridine, 2'-O-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, methyl ester of uridine 5-oxyacetic acid, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, or 5-(isopentenylaminomethyl)2'-O-methyluridine, the combination according to claim 6. **Claim 8** At least one modified nucleotide is a sugar-modified nucleotide, preferably a 2' ribose-modified RNA nucleotide, More preferably, it is a 2'-O-methylated RNA nucleotide, The 2'-O-methylated RNA nucleotide is most preferably selected from 2'-O-methylated guanosine (Gm), 2'-O-methylated uracil (Um), 2'-O-methylated adenosine (Am), 2'-O-methylated cytosine (Cm), or a 2'-O-methylated analog of any of these nucleotides, the combination according to claim 7.

9. The nucleic acid of the second component contains at least one or more trinucleotide M-X-Y motifs, wherein M is selected from Gm, Um, or Am, preferably M is Gm, wherein X is selected from G, A, or U, preferably X is G, wherein Y is selected from G, A, U, C, or dihydrouridine, preferably Y is C, and / or, the nucleic acid of the second component contains or consists of a nucleic acid sequence according to formula I N Ｗ -M-X-Y-N Ｚ (Formula I) wherein N is independently selected from G, A, U, C, Gm, Am, Um, Cm, or a modified nucleotide, wherein W is 0 or an integer from 1 to 15, wherein Z is 0 or an integer from 1 to 15, wherein M, X, and Y are selected as defined above, optionally, the nucleic acid of the second component contains or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I, and each of the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid sequences according to formula I is identical to each other or independently selected, and / or, the nucleic acid of the second component contains a 5'-end lacking a triphosphate group, the combination according to any one of claims 6-8.

10. The nucleic acid of the second component has a length of about 3 to about 50 nucleotides, about 5 to about 25 nucleotides, about 5 to about 15, or about 5 to about 10 nucleotides, preferably about 5 to about 15 nucleotides, optionally, the nucleic acid of the second component has a length of 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, or 13 nucleotides, preferably 9 nucleotides, the combination according to any one of claims 6-9.

11. The nucleic acid of the second component is a single-stranded oligonucleotide, ​ The combination according to any one of claims 6 to 10, wherein preferably the single-stranded oligonucleotide is a single-stranded RNA oligonucleotide.

12. The nucleic acid of the second component comprises, or consists of, a nucleic acid sequence derived from bacterial tRNA, preferably bacterial tRNA Ｔｙｒ and Optionally, wherein the nucleic acid sequence is a bacterial tRNA Ｔｙｒ , preferably a bacterial tRNA Ｔｙｒ D-loop, most preferably E. coli tRNA Ｔｙｒ D-loop, or a combination according to any one of claims 6 to 11, which is derived therefrom.

13. the nucleic acid of the second component is selected from the group consisting of nucleic acid sequences of SEQ ID NOs: 85 to 212, or is identical to a fragment of any of these sequences, or comprises a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of these nucleic acid sequences, preferably, the nucleic acid of the second component is selected from the group consisting of nucleic acid sequences of SEQ ID NOs: 85 to 87, 149 to 212, or is identical to a fragment of any of these sequences, or comprises a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of these nucleic acid sequences, more preferably, the nucleic acid of the second component is a nucleic acid sequence according to 5'-GAG CGMG CCA-3' (SEQ ID NO: 85), or is identical to a fragment thereof, or comprises a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or consists of that nucleic acid sequence, the combination according to any one of claims 6 to 12.

14. the coding RNA is selected from mRNA, self-replicating RNA, circular RNA, viral RNA, or replicon RNA, preferably selected from mRNA, the coding RNA or mRNA comprises at least one coding sequence encoding at least one peptide or protein, Preferably, compared to expressing at least one encoded peptide or protein of the coding RNA or mRNA without combining with at least one antagonist of at least one RNA-sensing pattern recognition receptor of the second component, upon administration to a cell, tissue or organism, by combining with at least one antagonist of at least one RNA-sensing receptor of the second component, the expression of at least one encoded peptide or protein of the coding RNA or mRNA is increased or extended, wherein the at least one peptide or protein is or is derived from a therapeutic peptide or a therapeutic protein, the therapeutic peptide or the therapeutic protein is preferably an antibody, cytokine, receptor, receptor agonist, receptor antagonist, binding protein, CRISPR-related endonuclease, chaperone, transporter protein, ion channel, membrane protein, secreted protein, transcription factor, enzyme, peptide hormone or protein hormone, growth factor, structural protein, cytoplasmic protein, cytoskeletal protein, viral antigen, bacterial antigen, protozoal antigen, allergen, tumor antigen, or a fragment, or variant, combination of any of these, or preferably is derived therefrom, the at least one coding sequence is more preferably a codon-modified coding sequence, the at least one codon-modified coding sequence is most preferably selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage frequency-conforming coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof, the combination according to any one of claims 1 to 13. **Claim 15** at least one therapeutic RNA of the first component, preferably mRNA, contains a 5'-cap structure, preferably, the 5'-cap structure is a cap0, cap1, cap2, modified cap0 or modified cap1 structure, more preferably, the 5'-cap structure is a cap1 structure, the combination according to any one of claims 1 to 14. **Claim 16** at least one therapeutic RNA of the first component contains at least one modified nucleotide or modified nucleotide analog, The combination according to any one of claims 1 to 15, wherein the at least one modified nucleotide is preferably selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and / or 5-methoxyuridine.

17. At least one therapeutic RNA of the first component, preferably mRNA, comprises at least one poly(A) sequence, and / or at least one poly(C) sequence, and / or at least one histone stem-loop sequence / structure. Preferably, the poly(A) sequence is located at the 3'-end of the therapeutic RNA, and / or the 3'-end of the RNA consists of a poly(A) sequence terminated with A nucleotides. And / or At least one therapeutic RNA of the first component, preferably mRNA, comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. The at least one heterologous 3'-UTR preferably comprises a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or a nucleic acid sequence derived from a homolog, fragment, or variant of any of these genes. Or The at least one heterologous 5'-UTR preferably comprises a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUB4B, and UBQLN2, or a nucleic acid sequence derived from a homolog, fragment, or variant of any of these genes. The combination according to any one of claims 1 to 16.

18. The LNP comprises (i) at least one cationic lipid (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (iii) at least one steroid or steroid analog, preferably cholesterol, and (iv) at least one PEG lipid and Preferably, (i) to (iv) are in a molar ratio of about 20 to 60% cationic lipid; 5 to 25% neutral lipid; 25 to 55% sterol; 0.5 to 15% PEG lipid. The combination according to any one of claims 1 to 17.

19. A pharmaceutical composition comprising or consisting of the combination according to any one of claims 1 to 18 and optionally at least one pharmaceutically acceptable carrier, wherein at least one therapeutic RNA and at least one antagonist are more preferably co-formulated to increase the probability that both are present in one particle and to ensure that at least one therapeutic RNA and at least one antagonist are taken up by the same cell.

20. The molar ratio of at least one antagonist, preferably a nucleic acid, to at least one therapeutic RNA is in the range of about 1:1 to about 100:1, or in the range of about 20:1 to about 80:1, and / or The weight-to-weight ratio of at least one antagonist, preferably a nucleic acid, to at least one therapeutic RNA is in the range of about 1:1 to about 1:30, or in the range of about 1:2 to about 1:10, for the pharmaceutical composition according to claim 19.

21. Administration of the composition to a cell, tissue, or organism results in essentially the same or at least equivalent activity of the therapeutic RNA as compared to administration of the corresponding therapeutic RNA alone, or Administration of the composition to a cell, tissue, or organism results in an increase in the activity of the therapeutic RNA, for example as compared to administration of the corresponding therapeutic RNA alone, wherein the activity of the therapeutic RNA is preferably the expression of the encoded peptide or protein, more preferably the expression of the protein, for the pharmaceutical composition according to claim 19 or 20.

22. Administration of the composition to a cell, tissue, or organism results in a reduction in (innate) immune activation as compared to administration of the corresponding therapeutic RNA alone, for the pharmaceutical composition according to any one of claims 19 to 21.

23. The combination according to any one of claims 1 to 18, or the pharmaceutical composition according to any one of claims 19 to 22, for use as a medicament.

24. For use in chronic treatment, or For use in the treatment or prevention of an infectious disease, preferably a viral infection, a bacterial infection, or a protozoal infection, or For use in the treatment or prevention of a tumor disease or a disorder associated with such a tumor disease, or For use in the treatment or prevention of a genetic disorder or condition, or For use in the treatment or prevention of protein or enzyme deficiency, The combination according to any one of claims 1 to 18, or the pharmaceutical composition according to any one of claims 19 to 22.

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