Novel dsrna binding ligands and their use in affinity chromatography

Abstract

The present invention relates to a novel affinity separation matrix that comprises novel binding ligands for double stranded RNA (dsRNA) and the use of said affinity separation matrix for purifying ribonucleic acid preparations. The present invention further relates to a method of purifying ribonucleic acid preparations and a kit for use in a method of purifying ribonucleic acid preparations, implementing the novel affinity separation matrix. The present disclosure also relates to a method of production of a single-stranded RNA (ssRNA) preparation.

Description

[0001] NOVEL dsRNA BINDING LIGANDS AND THEIR USE IN AFFINITY CHROMATOGRAPHY

[0002] TECHNICAL FIELD

[0003] The present invention relates to a novel affinity separation matrix that comprises novel binding ligands for double stranded RNA (dsRNA) and the use of said affinity separation matrix for purifying ribonucleic acid preparations. The present invention further relates to a method of purifying ribonucleic acid preparations and a kit for use in a method of purifying ribonucleic acid preparations, implementing the novel affinity separation matrix. The present disclosure also relates to a method of production of a single-stranded RNA (ssRNA) preparation.

[0004] BACKGROUND OF THE INVENTION

[0005] Preparations of single-stranded RNA synthesized by in vitro transcription (IVT) often contain significant amounts of aberrant products, such as nucleic contaminants (e.g., DNA template or dsRNA) and protein contaminants (e.g., T7 RNA polymerase). Methods for removal of DNA contaminants (e.g., digestion of DNA by DNases) or protein contaminants (e.g., via phenolchloroform precipitation) from RNA preparations are well-known in the art. Synthetically produced messenger RNA (mRNA) is exploited for therapeutic applications. Since dsRNA has been described to induce inflammatory cytokines and activate effector enzymes leading to inhibition of protein synthesis, it is desirable a critical impurity and therefore, it is to remove dsRNA in the development of RNA therapeutics that are based on in vitro transcribed mRNA (IVT mRNA).

[0006] WO 2017 / 182524 A1 discloses methods for providing ssRNA comprising contacting an IVT RNA preparation with a cellulose material under conditions, which allow binding of dsRNA to the cellulose material, and separating the ssRNA from the cellulose material.

[0007] WO 2013 / 102203 A1 discloses an enzyme-based method using E. coli RNase III enzyme, which specifically hydrolyzes dsRNA but not ssRNA to remove dsRNA contaminants from IVT mRNA preparations.

[0008] The known methods for producing ssRNA have turned out to be associated with several shortcomings such as impurity of the ssRNA preparations. There is thus still a strong need in the art to provide novel means and methods for purifying RNA preparations. The present invention addresses this need and provides a novel affinity separation matrix that comprises novel binding ligands for double stranded RNA (dsRNA).

[0009] SUMMARY OF THE INVENTION The novel affinity separation matrix provided by the present invention allows purifying ribonucleic acid preparations based on novel binding ligands that exhibit a high specificity for dsRNA contaminations from ribonucleic acid preparations.

[0010] In particular, the present invention provides the following items 1 to 15, without being specifically limited thereto:

[0011] [1] An affinity separation matrix for purifying a ribonucleic acid preparation comprising a binding ligand for double stranded RNA (dsRNA), wherein the dsRNA binding ligand comprises at least one dsRNA binding domain comprising an amino acid sequence of any one of SEQ ID NOs: 1-6, or a sequence with at least 90 % identity thereto.

[0012] [2] The affinity separation matrix according to item 1 wherein the dsRNA binding ligand comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4, or a sequence with at least 90 % identity thereto.

[0013] [3] The affinity separation matrix according to item 1 or 2, wherein the dsRNA binding ligand comprises the dsRNA binding domains comprising: a) the amino acid sequences of SEQ ID NOs: 1 and 4, or sequences with at least 90 % identity thereto; or b) the amino acid sequences of SEQ ID NOs: 4 and 5, or sequences with at least 90 % identity thereto; or c) the amino acid sequences of SEQ ID NOs: 3 and 4, or sequences with at least 90 % identity thereto.

[0014] [4] The affinity separation matrix according to any one of [1] to [3], wherein the dsRNA binding ligand has more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, preferably wherein the dsRNA binding ligand has more than 10-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions, more preferably wherein the dsRNA binding ligand has more than 50-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions.

[0015] [5] The affinity separation matrix of any one of [1] to [4], wherein the dsRNA binding ligand has a binding affinity for dsRNA of about 200 nM or less, in particular wherein the dsRNA binding ligand has a binding affinity of about 200 nM or less for dsRNA with a length of about 40 bp, or with a length of about at least 40 bp, such as a length of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 (or more) bp.

[0016] [6] The affinity separation matrix of any one of [1] to [5], wherein the dsRNA binding ligand comprises at least two dsRNA binding domains, which are linked by a linker, preferably wherein the linker is a peptide linker, in particular wherein the peptide linker has a length of at least 5 amino acid residues. [7] The affinity separation matrix according to any one of [1] to [6], wherein the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 7 to 9, or an amino acid sequence with at least 90 % identity thereto.

[0017] [8] The affinity separation matrix according to any one of [1] to [7], wherein the dsRNA binding ligand is fused to a Protein A domain or a Protein A-like protein, preferably wherein the Protein A domain or the Protein A-like protein lacks detectable binding to dsRNA.

[0018] [9] A fusion protein comprising the dsRNA binding ligand according to any one of [1] to [7] and a Protein A domain or Protein A-like protein, preferably wherein the Protein A-like protein lacks detectable binding affinity to dsRNA, preferably wherein the Protein A domain or Protein A-like protein has at least 85 % amino acid sequence identity with a sequence of any of SEQ ID NO: 20, 31 , or 33.

[0019]

[0010] The fusion protein according to [9], wherein the fusion protein comprises an amino acid sequence with at least 90 % amino acid sequence identity to any one of SEQ ID NOs: 21-30.

[0020]

[0011] A method of purifying a ribonucleic acid preparation, comprising: a) providing a ribonucleic acid preparation containing single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), b) contacting the ribonucleic acid preparation of a) with the affinity separation matrix of any one of [1] to [8], or the fusion protein of [9] or

[0010] , to capture the dsRNA, c) separating the ssRNA from the captured dsRNA.

[0021] In various embodiments of the method of

[0011] , the contacting of the ribonucleic acid preparation with the affinity separation matrix comprises applying the ribonucleic acid preparation to a solid phase comprising the affinity separation matrix of any one of [1] to [8], or the fusion protein of

[0022] [9] or

[0010] ,

[0023]

[0012] Use of the affinity separation matrix of any one of [1] to [8], or the fusion protein of [9] or

[0024]

[0010] , in affinity purification, in particular in affinity purification of a ribonucleic acid preparation.

[0025]

[0013] A kit comprising the affinity separation matrix of any one of [1] to [8], or the fusion protein of [9] or

[0010] , In preferred embodiments, the kit is for use in a method of purifying a ribonucleic acid preparation, wherein the ribonucleic acid preparation may be a ribonucleic acid preparation as described elsewhere herein.

[0026]

[0014] The affinity separation matrix of any one of [1] to [8], or the method of

[0011] , or the use of

[0012] , or the kit of

[0013] , wherein the ribonucleic acid preparation is an mRNA preparation or a ssRNA preparation, preferably wherein the mRNA is therapeutic mRNA or the ssRNA is inhibitory ssRNA, respectively, e.g., wherein the inhibitory ssRNA is therapeutic inhibitory ssRNA.

[0027]

[0015] A method of production of a single-stranded RNA (ssRNA) preparation, the method comprising:

[0028] (i) providing a preparation that contains double-stranded RNA (dsRNA) and ssRNA; (ii) providing the affinity separation matrix of any one of [1] to [8], or the fusion protein of [9] or

[0029]

[0010] ;

[0030] (iii) contacting the preparation of (i) with the affinity separation matrix or the fusion protein of

[0031] (11) under conditions that allow capturing of dsRNA by the dsRNA binding ligand;

[0032] (iv) separating the complex of the dsRNA bound to the affinity separation matrix or the fusion protein from said preparation; and

[0033] (v) obtaining the ssRNA preparation.

[0034] This summary of the invention is not limiting, and further aspects and embodiments as well as advantages of the present invention will become apparent from the following detailed description, examples, and drawings.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides a novel affinity separation matrix having specific binding affinity for dsRNA. The novel affinity separation matrix is particularly advantageous because affinity ligands for dsRNA allow precise purification (capturing) of dsRNA in ribonucleic acid preparations, for example in affinity chromatography. Any polypeptide selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or an amino acid sequence with at least 90 % identity of any one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, bind to dsRNA and are suitable for example for the removal of contaminant dsRNA from a ribonucleic acid preparation, for example an mRNA or therapeutic ssRNA preparation.

[0037] Before the present disclosure is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects and embodiments only and is not intended to limit the scope of the present invention, which is reflected by the appended items. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This includes a skilled person working in the field of protein engineering and purification, but also including a skilled person working in the field of developing new specific binding molecules for dsRNA for use in technical applications, for example for use as affinity ligands in affinity chromatography.

[0038] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations, Leuenberger, H.G.W., Nagel, B., and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughout this specification and the items, which follow, unless the context requires otherwise, the word “comprise”, and variants such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The terms “comprise(s)” or “comprising” may encompass a limitation to “consists of’ or “consisting of”, should such a limitation be necessary for any reason and to any extent.

[0039] In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. As used in this specification, the singular forms “a”, “an”, and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. Further, as used herein, the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a number or a numerical range, it modifies that number or that range, respectively, by extending the boundaries above and below the numerical value(s) set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 %.

[0040] Several documents, for example patents, patent applications, scientific publications, manufacturer's specifications, instructions, UniProt Accession Numbers, may be cited throughout the present specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein may be characterized as being “incorporated by reference" . In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0041] All sequences referred to herein are disclosed in the attached sequence listing that with its whole content and disclosure, forms part of the disclosure content of the present specification.

[0042] General definitions of important terms used herein

[0043] The term “nucleic acid”, “nucleotide”, and “polynucleotide” may be used interchangeably herein and encompass any nucleic acid molecule having a nucleotide sequence comprising purine and pyrimidine bases which are comprised by said nucleic molecule or sequence, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid can include a single or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases read from the 5’ to the 3’ end and include double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), double stranded RNA (dsRNA), genomic DNA, cDNA, cRNA, recombinant DNA or recombinant RNA and derivatives thereof, such as those containing modified backbones. In some embodiments, the polynucleotide of the present invention can be composed of any polyribonucleotides, which may be unmodified or modified RNA. Polynucleotides according to the invention can be construed in different ways (e.g. by chemical synthesis, by gene cloning, etc.) and can take various forms (e.g. linear or branched, single or double stranded, or a hybrid thereof, primes, probes etc.) The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.

[0044] The term “ribonucleic acid preparation” as described herein refers to a ssRNA-containing preparation, such as mRNA synthesized via in vitro transcription (IVT), that is to be purified from contaminant dsRNA. A ssRNA is a polymeric strand of contiguous ribonucleotides. A dsRNA is comprised of two polymeric strands of contiguous ribonucleotides bound to each other through complementary ribonucleotide base pairing. ssRNA includes, without limitation, mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), microRNA (miRNA), short hairpin RNA (shRNA), and non-coding RNA (ncRNA). ssRNAs as described herein, may form intramolecular secondary structures and may be double-stranded or partially double-stranded.

[0045] The term “amino acid sequence identity” refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine the sequence identity, the sequence of a query protein is aligned to the sequence of a reference protein or polypeptide, for example, to the polypeptide of any one of SEQ ID NOs: 1-6. Methods for the sequence alignment are well known in the art. For example, for determining the extent of an amino acid sequence identity of an arbitrary polypeptide relative to the amino acid sequence of, for example, any one of SEQ ID NOs: 1-6, the SIM Local similarity program is preferably employed (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357), that is freely available. For multiple alignment analysis, ClustalW is preferably used (Thompson et al. (1994), Nucleic Acids Res., 22(22): 4673-4680).

[0046] The term “alkaline stability” or “caustic stability” refers to the ability of the binding protein for dsRNA to withstand alkaline conditions without significantly losing the ability to bind dsRNA. The skilled person in this field can easily test alkaline stability by incubating a binding protein for dsRNA with sodium hydroxide solutions, e.g., as described in Example 3, and subsequent testing of the binding affinity to dsRNA by routine experiments known to someone skilled in the art, for example, by chromatographic approaches.

[0047] The term “chromatography” refers herein to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules (e.g., dsRNA) from other molecules (e.g., ssRNA) in a ribonucleic acid preparation. The liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid matrix). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated.

[0048] The term “affinity chromatography” refers to a specific mode of chromatography in which a ligand (i.e., a binding protein for dsRNA) coupled to a stationary phase interacts with a molecule (i.e., dsRNA) in the mobile phase (the liquid sample), i.e., the ligand has a specific binding affinity for the molecule to be captured. As understood in the context of the invention, affinity chromatography involves the addition of a (liquid) ribonucleic acid preparation containing dsRNA to a stationary phase which comprises a chromatography binding ligand, such as a binding ligand for dsRNA. The terms “solid support” or “solid matrix” are used interchangeably for the stationary phase.

[0049] The terms “affinity matrix” or “affinity purification matrix” as used interchangeably herein, refer to a matrix, e.g., a chromatographic matrix, onto which an affinity ligand (e.g., a binding ligand for dsRNA) is attached. The attached affinity ligand (e.g., binding domain for dsRNA) is capable of specific binding to a molecule of interest (e.g., dsRNA) which is to be purified or separated or removed (captured) from a (liquid) sample or ribonucleic acid preparation. Accordingly, the terms “affinity separation matrix” and “affinity purification matrix” may be used interchangeably herein.

[0050] The term “affinity purification” or “affinity capturing” as used herein is related to a method of purifying (capturing) dsRNA from a ribonucleic acid preparation by binding dsRNA to a ligand for dsRNA that is immobilized to a matrix. Thereby, dsRNA is removed (captured) from the ribonucleic acid preparation, thereby purifying the ribonucleic acid preparation. As will be appreciated by a person of ordinary skill in the art upon review of the present specification, the method of purifying a ribonucleic acid preparation of the present invention is in particular an affinity chromatography method (of purifying a ribonucleic acid preparation). Accordingly, the “affinity separation matrix” (or “affinity purification matrix”) of the present invention may be considered as “affinity chromatography matrix”.

[0051] The term “binding ligand” as used herein refers to a dsRNA binding ligand, which comprises at least one dsRNA binding domain comprising an amino acid sequence of any one of SEQ ID NO: 1-6, or an amino acid sequence with at least 90 % identity to any one of SEQ ID NOs: 1- 6.. The dsRNA binding ligand can from part of, but is not limited to, a fusion protein comprising a Protein A domain or Protein A-like protein, as described elsewhere herein. By way of exhibiting binding affinity for dsRNA, the dsRNA binding ligand can be considered to include a dsRNA binding motif. The term “binding affinity” refers to the ability of a binding ligand of the present invention to a ribonucleic acid, in particular a ssRNA or a dsRNA, as described elsewhere herein and in accordance with the appended claims. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of biomolecular interactions. The binding affinity and dissociation constants can be measured quantitatively. Methods for determining binding affinities are well known to the skilled person and can be selected, for instance, from the following methods, which are well established in the art: surface plasmon resonance (SPR), Bio-layer interferometry (BLI), enzyme linked immunosorbent assay (ELISA), kinetic exclusion analysis (KinExA assay), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Typically, the dissociation constant KD is determined at temperatures in the range of 20°C and 30°C. If not specifically indicated otherwise, KD values recited herein are determined at room temperature by BLI. Room temperature can be considered 20-23 °C. In various embodiments of the present invention, the binding affinity for the binding ligand may be determined by the ForteBio - Octet QK384 blue BLI system or Sierra SPR-32 system (Bruker), at room temperature. As described herein, room temperature may in particular be considered to be a temperature of 20 °C.

[0052] The term “fusion protein” is defined as a protein which contains the (complete) amino acid sequences of any parts of the sequences of two or more originally separate natural or modified heterologous proteins or subunits of a protein or a composition of complete sequences or parts of the sequences of two or more originally separate natural or modified heterologous proteins or subunits of a protein. A fusion protein can be construed by genetic engineering approaches by fusing two or more genes, or parts thereof, that originally encode the two or more originally separate proteins or parts thereof. This results in a fusion protein with functional properties derived from each of the original proteins, subunits of the protein or parts thereof. Thus, a fusion protein may comprise a multimer of identical or different proteins which are expressed as a single, linear polypeptide or as a subunit thereof. Fusion proteins of the present invention include, but are not limited to, dsRNA binding ligands or dsRNA binding domains.

[0053] The term “protein” may be used interchangeably with the term “polypeptide” and refers to polymers of amino acids of any length typically for proteins or polypeptides of the present invention. These terms also include proteins that are post-translationally modified through reactions that include, but are not limited to, phosphorylation, glycosylation, acetylation, or for eukaryotic host cells also protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with the same or advantageous properties. The term “polypeptide” typically refers to a sequence of 10 or more amino acids linked by peptide bonds and the term “peptide” typically means sequences with up to 10 amino acids in length, unless dictated otherwhise. However, the terms may be used interchangeably with respect to, e.g., a peptide linker linking two dsRNA binding domains comprised by a dsRNA binding ligand of the invention. The same applies with regard to, e.g., a peptide linker linking a dsRNA binding ligand and a Protein A domain or Protein A-like protein, as described elsewhere herein.

[0054] Detailed description of the embodiments of the invention

[0055] The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect defined below may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature or features indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. The same applies with regard to features that are identified or described in the specification or the appended claims by using the term “in particular”, as exemplified by describing that a peptide linker in particular has a length of at least 5 amino acid residues. As used herein, the term “in particular” is not to be understood as limiting the feature or subject matter to which it refers and which is further specified by the term “in particular”.

[0056] The novel affinity separation matrix for purifying a ribonucleic acid preparation comprises a binding ligand, which exhibits specific binding affinity for double stranded RNA (dsRNA). The dsRNA binding ligand comprises at least one dsRNA binding domain comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6, or at least one amino acid sequence with at least 90 % sequence identity to any one of SEQ ID NOs: 1-6. The amino acid sequences according to SEQ ID NOs: 1-6, including amino acid sequence with at least 90 % sequence identity to any one of SEQ ID NOs: 1-6, are considered as dsRNA binding domains of the present invention.

[0057] The present invention encompasses novel dsRNA binding ligands comprising at least one dsRNA binding domain comprising an amino acid sequence of any one of SEQ ID NOs: 1-6, or a sequence with at least 90 % identity thereto.

[0058] In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 1 , or an amino acid with at least 90 % sequence identity to SEQ ID NO: 1 , including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 2, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0059] In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 3, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0060] In various preferred embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 4, including any of at least 90 %, 91 %, 92 %, 93 %,

[0061] 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0062] In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 5, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %,

[0063] 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0064] In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 6, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0065] The dsRNA binding ligand of the invention comprises at least one dsRNA binding domain, and may thus comprise only one single dsRNA binding domain. In preferred embodiments, the binding ligand for dsRNA comprises two or more dsRNA binding domains comprising at least two amino acid sequences of any of SEQ ID NOs: 1-6, or at least two amino acid sequences, each of which having at least 90 % sequence identity to any of SEQ ID NOs: 1-6.

[0066] In various embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising has at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 1-6.

[0067] In preferred embodiments, the binding ligand for dsRNA comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with at least 90 % sequence identity to SEQ ID NO: 4, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0068] In preferred embodiments of the present invention, the dsRNA binding ligand comprises at least two dsRNA binding domains, as described above. Accordingly, in preferred embodiments of the present invention, the binding ligand for dsRNA is a multimer composed of at least two dsRNA binding domains described herein.

[0069] The present invention encompasses novel dsRNA binding ligands comprises at least two dsRNA binding domains as described herein. Such multimeric forms are typically linked by a linker, preferably the linker is a peptide linker, in particular the peptide linker has a length of at least 5 amino acid residues. The peptide linker may or may not have a length of more than 10 amino acid residues. In such cases, the peptide linker may be considered a polypeptide linker. In more preferred embodiments, the peptide linker has a length of about 5 to about 100 amino acid residues, as described elsewhere herein.

[0070] It is preferred that such multimer dsRNA binding ligands of the invention comprising at least two dsRNA binding domains comprise a dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 4, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In such embodiments, the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid with at least 90 % sequence identity to SEQ ID NO: 4 may be an additional dsRNA binding domain or may be one of the at least two dsRNA binding domains. Preferably, the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid with at least 90 % sequence identity to SEQ ID NO: 4 is one of the at least two dsRNA binding domains.

[0071] In various embodiments, it may be preferred that a (multimeric) dsRNA binding ligand of the invention contains no more than two dsRNA binding domains of the dsRNA binding domains disclosed herein (“dimer”). More specifically, in such embodiments, the dsRNA binding ligand of the invention contains no dsRNA binding domain other than the two dsRNA binding domains (“dimer”). Accordingly, in particularly preferred embodiments of the invention, the (dimeric) dsRNA binding ligand comprises no more than two dsRNA binding domains, wherein one of the two dsRNA binding domains comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid with at least 90 % sequence identity to SEQ ID NO: 4, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the binding ligand for dsRNA as described herein comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences of SEQ ID NOs: 1 and 4. In various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences with at least 90 % sequence identity to SEQ ID NOs: 1 and 4, respectively. In various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequences of SEQ ID NOs: 1 and 4, respectively.

[0072] In some embodiments, the binding ligand for dsRNA as described herein comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences of SEQ ID NOs: 4 and 5. In various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences with at least 90 % sequence identity to SEQ ID NOs: 4 and 5, respectively. In various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequences of SEQ ID NOs: 4 and 5, respectively.

[0073] In some embodiments, the binding ligand for dsRNA as described herein comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences of SEQ ID NOs: 3 and 4. J n various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising the amino acid sequences with at least 90 %_sequence identity to SEQ ID NOs: 3 and 4, respectively. In various embodiments, the binding ligand for dsRNA comprises, or consists of, the dsRNA binding domain comprising at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

[0074] The affinity chromatography matrices of the present invention are based on dsRNA binding ligands comprising individual dsRNA binding domains, which in nature form part of a multidomain enzyme complex (RNA editing enzymes; ADAR (adenosine deaminase acting on RNA) proteins). Accordingly, as described herein, the present invention does not encompass dsRNA binding ligands that comprise all domains of a (multi-domain) RNA editing enzyme (“ADAR”), which is the (multi-domain) RNA editing enzyme to which a respective dsRNA binding domain disclosed herein belongs. The dsRNA binding domains disclosed herein may therefore be considered isolated dsRNA binding domains.

[0075] One advantage of the herein disclosed binding ligands for dsRNA is the important functional characteristic that it binds specifically to dsRNA. This is of particular advantage in capturing of contaminant dsRNA in the purification process of ribonucleic acid preparations, for example in the process of production of therapeutic mRNA or ssRNA preparations. Example 4 shows the depletion of dsRNA from ribonucleic acid preparations.

[0076] The binding ligand for dsRNA is functionally characterized by at least, or more than, 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, as shown in Example 2. In some embodiments, the dsRNA binding ligand has at least, or more than, 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, as determined by BLI or SPR. Further described herein, and encompassed by the present invention, is that the dsRNA binding ligand has at least, or more than, 5-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, as determined by BLI or SPR.

[0077] In various embodiments, the dsRNA binding ligand has at least or more than (5-fold or) 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, wherein the length of the dsRNA and ssRNA corresponds to each other. For example, in preferred embodiments, the dsRNA binding ligand has at least, or more than, (5- fold or) 10-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions. Typically, the same conditions mean the same experimental conditions, and typically include the same or corresponding length of the dsRNA and / or ssRNA used for determining or comparing binding affinities of the dsRNA binding ligands of the present invention. In preferred embodiments, the binding affinity for 40 nt ssRNA (under the same conditions as for 40 bp dsRNA) is not detectable. In other preferred embodiments, the dsRNA binding ligand has more than 50-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions. In further preferred embodiments, the dsRNA binding ligand has more than 100-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions.

[0078] Ribonucleic acid preparations can contain heterogeneous dsRNA impurities. For example, in vitro transcribed (“IVT”) mRNA typically contains heterogeneous dsRNA impurities. IVT mRNA length varies widely, ranging from less than 50 nucleotides (nt) to up to more than 9,000 nt, depending on the intended application. Accordingly, while prevalent sizes of dsRNA impurities in IVT mRNA product preparations may be in the range of about 40 - 250 bp, longer duplexes (hundreds to several thousand bp) and very short duplexes (<40 bp) can also be present, depending on IVT setup and downstream handling.

[0079] As described herein, dsRNA impurities in ribonucleic acid preparations according to the present invention include, without being limited thereto, dsRNA with a length ranging from less than 50 bp to up to more than 9,000 bp. As demonstrated in the Examples, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising of the invention comprising the dsRNA binding ligands, bind to different sizes of dsRNA, including short and longer duplexes.

[0080] The dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length of about 40 bp or less and longer duplexes comprising hundreds or even more than 1 ,000 bp.

[0081] In various embodiments, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length ranging from about 40 bp or less to about 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1 ,000 bp. The length of the dsRNAs may be ranging from about 40 bp or less to about 250 bp, or from about 40 bp or less to about 800 bp.

[0082] In various embodiments, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length ranging from about 40 bp or less to >1 ,000 bp. In various embodiments, the dsRNAs have a length up to about 2,000 bp, or up to about 3,000 bp. In various embodiments, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length ranging from about 40 bp or less to about 2,000 bp, or up to about 3,000 bp.

[0083] In various embodiments, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length of >3,000 bp. In various embodiments, the dsRNAs have a length of up to about 4,000 bp, or up to about 5,000 bp.

[0084] In various embodiments, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, bind to dsRNAs with a length of >5,000 bp. In various embodiments, the dsRNAs have a length of up to about 8,000 bp, or a length of up to about 9,000 bp, or even >9,000 bp.

[0085] As further described herein, in various embodiments, short dsRNAs may be considered to have a length of about 40 bp or less to about 50 bp, in particular about 30 bp to about 50 bp, and long dsRNAs may be considered to have a length ranging from about 500 bp to about 1 ,000 bp, or >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp.

[0086] As further described herein, the length of dsRNA impurities includes lengths equivalent to the full-length mRNA transcript.

[0087] The functional characteristics concerning binding affinity for dsRNA of the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, apply to any of the dsRNA lengths described above and elsewhere herein. In various embodiments, the dsRNA binding ligand has a binding affinity for dsRNA of 200 nM or less. In preferred embodiments, the dsRNA binding protein has a binding affinity of 200 nM or less for dsRNA with a length of about 40 bp. In other embodiments, the dsRNA binding ligand, in particular an affinity separation matrix comprising the dsRNA binding ligand, has a binding affinity of 200 nM or less for dsRNA with a length of at least 40 bp (e.g. 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 (or more) bp. In preferred embodiments, the dsRNA binding protein has a binding affinity of 100 nM or less for dsRNA with a length of about 40 bp. In other embodiments, the dsRNA binding protein, in particular an affinity separation matrix comprising the dsRNA binding ligand, has a binding affinity of 100 nM or less for dsRNA with a length of at least 40 bp (e.g. 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 (or more) bp.

[0088] The binding affinity of 200 nM or less, or 100 nM or less, for dsRNA applies to any dsRNA length disclosed herein, in particular, but not limited to, any length ranging from about 40 bp (or less) to >1 ,000 bp, specifically a length up to about 2,000 bp, or up to about 3,000 bp. In various embodiments, the dsRNAs has a length ranging from about 500 bp to >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp.

[0089] In some embodiments, the dsRNA binding ligand, in particular an affinity separation matrix comprising the dsRNA binding ligand, has a binding affinity for 200 bp dsRNA of less than 50 nM.

[0090] As described elsewhere herein, in some embodiments of the invention, two or more binding ligands for dsRNA of the invention may be linked to each other. In such embodiments, it is preferred that no more than two binding ligands for dsRNA of the invention are linked to each other.

[0091] The binding affinities described herein above apply to monomers and multimers, i.e. , apply to dsRNA binding ligands comprising only one dsRNA binding domain of the invention (“monomer”), as well as dsRNA binding ligands comprising two or more dsRNA binding domains of the invention. The binding affinities described herein above apply in particular to dsRNA binding ligands comprising two dsRNA binding domains of the invention (“dimer”).

[0092] As described elsewhere herein, in certain embodiments of the invention, the binding ligand for dsRNA is a multimer that comprises (at least) two dsRNA binding domains. These two dsRNA binding domains are linked to each other. In preferred embodiments, the two, three, or four dsRNA binding domains are linked to each other by a linker. In preferred embodiments, the linker is a peptide linker. In preferred embodiments, the peptide linker has a length of at least 5 amino acid residues. In more preferred embodiments, the peptide linker has a length of about 5 to about 100 amino acid residues. In various embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 7 to 9. In some embodiments, the peptide linker comprises an amino acid sequence with at least 90 % sequence identity to any one of SEQ ID NOs: 7 to 9, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any of SEQ ID NOs: 7 to 9. In preferred embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence with at least 90 % sequence identity to SEQ ID NO: 7, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0093] The peptide linkers described herein for linking (at least) two dsRNA binding domains to each other, in particular a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 7 to 9, or comprising an amino acid sequence with at least 90 % sequence identity to any of SEQ ID NOs: 7 to 9, are preferably used to link the dsRNA binding domains comprising a) the amino acid sequences of SEQ ID NOs: 1 and 4, or sequences with at least 90 % identity thereto; b) the amino acid sequences of SEQ ID NOs: 4 and 5, or sequences with at least 90 % identity thereto; or c) the amino acid sequences of SEQ ID NOs: 3 and 4, or sequences with at least 90 % identity thereto, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1 and 4, SEQ ID NO: 4 and 5, or SEQ ID NO: 3 and 4, respectively.

[0094] Similar to two or more dsRNA binding domains being linked to each other via a linker, in case of two or more binding ligands for dsRNA of the invention being linked to each other, such two or more dsRNA binding ligands may also be linked to each other via a linker, in particular via a peptide linker. Such a peptide linker may have a length of at least 5 amino acid residues. More specifically, such a linker may have a length of about 5 to about 100 amino acid residues. In some embodiments, the peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 7 to 9, or may comprise an amino acid sequence with at least 90 % sequence identity to any of SEQ ID NOs: 7 to 9, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any of SEQ ID NOs: 7 to 9.

[0095] Multimers of the dsRNA binding ligand of the invention are typically generated artificially, generally by recombinant DNA technology well known to a skilled person. In various embodiments, the multimer is a homo-multimer, e.g. the amino acid sequences of the binding ligand for dsRNA comprising the dsRNA binding domains are identical. In preferred embodiments, the multimer is a hetero-multimer, e.g., the amino acid sequences of the binding ligand for dsRNA comprising the dsRNA binding domains are different, as reflected by SEQ ID NOs: 10, 16, or 19. In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 10, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0096] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 11 , or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 11, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0097] In some embodiments, the peptide linker comprised by SEQ ID NO: 10 or 11, which is the linker of SEQ ID NO: 7 or 8, respectively, can be replaced with the peptide linker of SEQ ID NO: 9.

[0098] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 12, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0099] In some embodiments, the peptide linker comprised by SEQ ID NO: 12, which is the linker of SEQ ID NO: 8, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 9.

[0100] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 13, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0101] In some embodiments, the peptide linker comprised by SEQ ID NO: 13, which is the linker of SEQ ID NO: 9, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 8.

[0102] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 14, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0103] In some embodiments, the peptide linker comprised by SEQ ID NO: 14, which is the linker of SEQ ID NO: 9, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 8. In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 15, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15.

[0104] In some embodiments, the peptide linker comprised by SEQ ID NO: 15, which is the linker of SEQ ID NO: 8, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 9.

[0105] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 16, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0106] In some embodiments, the peptide linker comprised by SEQ ID NO: 16, which is the linker of SEQ ID NO: 7, can be replaced with the peptide linker of SEQ ID NO: 8 or SEQ ID NO: 9.

[0107] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 17, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0108] In some embodiments, the peptide linker comprised by SEQ ID NO: 17, which is the linker of SEQ ID NO: 9, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 8.

[0109] In various embodiments, the dsRNA binding ligand comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 18, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0110] In some embodiments, the peptide linker comprised by SEQ ID NO: 18, which is the linker of SEQ ID NO: 8, can be replaced with the peptide linker of SEQ ID NO: 7 or SEQ ID NO: 9.

[0111] In various embodiments, the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 19, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19.

[0112] In some embodiments, the peptide linker comprised by SEQ ID NO: 19, which is the linker of SEQ ID NO: 7, can be replaced with the peptide linker of SEQ ID NO: 8 or SEQ ID NO: 9. As described herein, a dsRNA binding domain, by virtue of the dsRNA binding ligand exhibiting binding affinity to dsRNA, can be considered to comprise a dsRNA binding motif (dsRBM). The dsRBM of the dsRNA binding domain may be a apppa fold. In preferred embodiments, the dsRNA binding domain may ne considered to comprise a dsRBM, which is a OiPiP2P3C(2 fold.

[0113] Surprisingly, the dsRNA binding ligands comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6 as described herein have the functional characteristic of binding with high specificity (and affinity) to dsRNA. The functional characteristic is considered to be the result of a proper (uniform) folding of the dsRNA binding ligand of the present invention, which could not have been expected by the one of ordinary skill in the art and which is thus considered surprising.

[0114] Fusion proteins. In some embodiments, the invention refers to a fusion protein comprising at least one dsRNA binding ligand of the present invention and at least one a Protein A domain or a Protein A-like domain (protein), preferably wherein the Protein A domain thereof or a Protein A-like domain lacks (or has no) detectable binding affinity to dsRNA. Further, the Protein A-like domain (protein) lacks (or has no) detectable binding affinity to immunoglobulin (IgG). Such embodiments encompass multimers of the dsRNA binding domains of the present invention.

[0115] Accordingly, in some embodiments, the invention refers to a fusion protein comprising at least one dsRNA binding ligand of the present invention and a Protein A domain or Protein A-like domain, preferably wherein the Protein A domain variants thereof or Protein A-like domain lacks detectable binding affinity to dsRNA.

[0116] The present invention provides a fusion protein comprising a double-stranded RNA (dsRNA) binding ligand as described herein, wherein the dsRNA binding ligand comprises at least one dsRNA binding domain comprising an amino acid sequence of any one of SEQ ID NOs: 1-6, or a sequence with at least 90 % identity to any one of SEQ ID NOs: 1-6, and a Protein A or Protein A-like protein, optionally wherein the Protein A-like protein lacks detectable binding affinity to dsRNA. The 90 % sequence identity to any one of SEQ ID NOs: 1-6 includes any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-6. As described elsewhere herein, the dsRNA binding ligand preferably comprises the dsRNA binding domain comprising the amino acid sequence of SEQ ID NO: 4, or a sequence with at least 90 % identity thereto, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. Further, as described elsewhere herein, in preferred embodiments, the dsRNA binding ligand comprises the dsRNA binding domains comprising: a) the amino acid sequences of SEQ ID NOs: 1 and 4, or sequences with at least 90 % identity thereto; b) the amino acid sequences of SEQ ID NOs: 4 and 5, or sequences with at least 90 % identity thereto; or c) the amino acid sequences of SEQ ID NOs: 3 and 4, or sequences with at least 90 % identity thereto, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any of SEQ ID NOs: 1 and 4, any of SEQ ID NOs: 4 and 5, and any of SEQ ID NOs: 3 and 4, respectively.

[0117] As described herein, the fusion protein has more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, preferably wherein the fusion protein has more than 10-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions.

[0118] In some embodiments, the fusion protein has more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, preferably wherein the fusion protein has more than 10-fold binding affinity for about at least 40 bp dsRNA as compared to the binding affinity for about at least 40 nt ssRNA under the same conditions. In some embodiments, the fusion protein has more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, preferably wherein the fusion protein has more than 10-fold binding affinity for at least 40, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, respectively, or more, bp dsRNA as compared to the binding affinity for about at least 40, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000, respectively, or more, nt ssRNA under the same conditions. The functional characteristics concerning a more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions apply to fusion proteins of the invention binding to dsRNA of any lengths as described above and elsewhere herein. The same applies with regard to a more than 50-fold binding affinity, and a more than 100-fold binding affinity, respectively, for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions, described elsewhere herein.

[0119] In various embodiments, the more than 10-fold, more than 50-fold, and more than 100-fold, respectively, binding affinity binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions applies to the binding affinity of fusion proteins of the invention binding to dsRNA having any length ranging from about 40 bp (or less) to >1 ,000 bp, specifically a length up to about 2,000 bp, or up to about 3,000 bp. In various embodiments, the dsRNAs has a length ranging from about 500 bp to >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp.

[0120] As further described herein, the fusion protein has a binding affinity for dsRNA of about 200 nM or less, in particular the fusion protein has a binding affinity of about 200 nM or less for dsRNA with a length of about 40 bp.

[0121] In some embodiments, the fusion protein has a binding affinity for dsRNA of about 100 nM or less, in particular the fusion protein has a binding affinity of about 100 nM or less for dsRNA with a length of about at least 40 bp. In some embodiments, the fusion protein has a binding affinity for dsRNA of about 200 nM or less, in particular the fusion protein has a binding affinity of about 200 nM or less for dsRNA with a length of about at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 or more bp. In some embodiments, the fusion protein has a binding affinity for dsRNA of about 100 nM or less, in particular the fusion protein has a binding affinity of about 100 nM or less for dsRNA with a length of about at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 or more bp. The functional characteristics concerning a binding affinity for dsRNA of the fusion protein of about 200 nM or less, or at least about 100 nM or less, apply to fusion proteins of the invention binding to dsRNA of any lengths as described above and elsewhere herein. In various embodiments, the binding affinity for dsRNA of the fusion protein of about 200 nM or less, or at least about 100 nM or less, applies to the binding affinity of fusion proteins of the invention binding to dsRNA having any length ranging from about 40 bp (or less) to >1 ,000 bp, specifically a length up to about 2,000 bp, or up to about 3,000 bp. In various embodiments, the dsRNAs has a length ranging from about 500 bp to >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp. In some embodiments, the fusion protein has a binding affinity for 200 bp dsRNA of less than 50 nM.

[0122] As further described herein, the fusion protein preferably comprises at least two dsRNA binding domains, which are linked by a linker, preferably wherein the linker is a peptide linker, in particular wherein the peptide linker has a length of at least 5 amino acid residues, as described elsewhere herein. The peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 7 to 9, or an amino acid sequence with at least 90 % identity thereto, as described elsewhere herein. Preferably the Protein A-like protein lacks detectable binding affinity to dsRNA.

[0123] As described elsewhere herein, the binding affinity to dsRNA can be determined by BLI, SPR or other suitable methods.

[0124] In the present invention, it is preferred that the Protein A domain or Protein A-like domain, which that lacks (or has no) detectable binding affinity to dsRNA is fused to the N-terminus of the dsRNA binding ligand. Accordingly, the present invention encompasses fusion proteins comprising at least one protein A domain or a Protein A-like domain as described herein at the N-terminus and at least one dsRNA binding ligand of the invention at the C-terminus of the fusion protein.

[0125] In preferred embodiments of the present invention, the Protein A-like domain comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence with at least 85 % sequence identity to the sequence of SEQ ID NO: 20, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In preferred embodiments of the present invention, the Protein A-like protein comprises the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 32, or an amino acid sequence with at least 85 % sequence identity to the sequence of SEQ ID NOs: 20 and 32, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20 and 32. In preferred embodiments, the amino acid sequence of SEQ ID NO: 20 described herein comprise a cysteine (Cys) residue at the position corresponding to position 12 of SEQ ID NO: 20.

[0126] The dimer of the Protein A-like protein may be homo-dimer or may be a hetero-dimer. It is preferred that the dimer of the Protein A-like protein is a hetero-dimer. Accordingly, the present invention encompasses a Protein A-like protein comprising or consisting of the amino acid sequence of SEQ ID NOs: 33 or 31 , or an amino acid sequence with at least 85 % sequence identity to the sequence of SEQ ID NOs: 33 or 31 , including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33 or 31. Further, the Protein A-like domain of SEQ ID NOs: 33 or 31 lack (or has no) detectable binding affinity to dsRNA. In preferred embodiments, the amino acid sequence of SEQ ID NOs: 33 or 31 described herein comprise a cysteine (Cys) residue at the position corresponding to position 12 of SEQ ID NOs: 33 or 31.

[0127] The present invention encompasses fusion proteins comprising a multimeric dsRNA binding ligand of the present invention.

[0128] In some preferred embodiments, the fusion protein comprises an amino acid sequence with at least 90 % amino acid sequence identity to any one of SEQ ID NOs: 21-30, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 21-30.

[0129] In some embodiments, the invention refers to a dsRNA binding ligand as described herein, or a multimeric dsRNA binding ligand as described herein, or a fusion protein described herein, which: (a) is capable of binding to dsRNA; and / or (b) has a binding affinity for 40 nucleotides of dsRNA of less than 1 pM, as determined by BLI.

[0130] In some embodiments, the invention refers to a fusion protein of the present invention, which has a binding affinity of less than 200 nM for dsRNA as described herein, in particular wherein the fusion protein has a binding affinity of about 200 nM or less for dsRNA with a length of about 40 bp, or with a length of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 ,000 (or more) bp. The functional characteristics concerning a binding affinity of the fusion protein for dsRNA of less than 200 nM apply to fusion proteins of the invention binding to dsRNA of any lengths as described above and elsewhere herein. In various embodiments, the binding affinity of the fusion protein for dsRNA of less than 200 nM applies to the binding affinity of fusion proteins of the invention binding to dsRNA having any length ranging from about 40 bp (or less) to >1 ,000 bp, specifically a length up to about 2,000 bp, or up to about 3,000 bp. In various embodiments, the dsRNAs has a length ranging from about 500 bp to >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp.

[0131] In some embodiments, the fusion protein of the present invention has a binding affinity of less than 100 nM for dsRNA as described herein. In some embodiments, the fusion protein of the present invention has a binding affinity of less than 50 nM for dsRNA as described herein. In preferred embodiments, the fusion protein of the present invention has a binding affinity of less than 30 nM for dsRNA as described herein. As will be appreciated by a person skilled in the art in view of the Examples, the dsRNA binding ligands disclosed herein, in particular an affinity separation matrix comprising the dsRNA binding ligands, and likewise a fusion protein of the present invention, can have a binding affinity of less than 50 nM for 200 bp dsRNA. According to another aspect, a method of purifying a ribonucleic acid preparation (by capturing the contaminant dsRNA) is provided. A ribonucleic acid preparation may contain ssRNA, preferably mRNA. As described herein, affinity chromatography, also referred to as affinity purification, makes use of specific binding interactions between molecules. Methods for immobilization of protein and methods for affinity chromatography are well-known in the field of protein purification and can be easily performed by a skilled person in the art using standard techniques and equipment. The method of purifying a ribonucleic acid preparation is provided, comprising the following steps, in particular to remove dsRNA contamination from the ribonucleic acid preparation: a) providing a ribonucleic acid preparation containing single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), b) contacting the ribonucleic acid preparation of a) with the affinity separation matrix as described herein to capture the dsRNA, c) separating the ssRNA from the captured dsRNA.

[0132] As will be appreciated by a person of ordinary skill in the art, in the methods of the present invention, the ribonucleic acid preparation may contain (or is considered or supposed to contain) dsRNA of any length, as described elsewhere herein. In particular, the ribonucleic acid preparation may contain (or is considered or supposed to contain) dsRNAs having any length ranging from about 40 bp (or less) to >1 ,000 bp, specifically a length up to about 2,000 bp, or up to about 3,000 bp. As further described herein, in various embodiments, the ribonucleic acid preparation may contain (or is considered or supposed to contain) dsRNAs having a length ranging from about 500 bp to about 1 ,000 bp, or >1 ,000 bp, e.g., up to about 2,000 bp, or up to about 3,000 bp.

[0133] Further encompassed are methods of the invention, wherein the ribonucleic acid preparation may contain (or is considered or supposed to contain) dsRNAs with a length of >3,000 bp, e.g., up to about 4,000 bp, or up to about 5,000 bp. Further encompassed are methods of the invention, wherein the ribonucleic acid preparation may contain (or is considered or supposed to contain) dsRNAs with a length of >5,000 bp, e.g., up to about 8,000 bp, or up to about 9,000 bp, or even >9,000 bp.

[0134] Affinity purification matrices suitable for the disclosed uses and methods are known to a person skilled in the art.

[0135] In various embodiments, the contacting of the ribonucleic acid preparation with the affinity separation matrix as described herein comprises applying the ribonucleic acid preparation to a solid phase comprising the affinity separation matrix according to the invention.

[0136] The terms “solid phase” or “solid support” as described herein are used interchangeably and refer to solid support matrices for affinity chromatography, which are known in the art and include, e.g., without being limited thereto, agarose and stabilized derivatives of agarose, cellulose or derivatives of cellulose, controlled pore glass, monolith, silica, zirconium oxide, titanium oxide, or synthetic polymers, and hydrogels or various compositions and combinations of the above. The formats for solid support matrices can be of any suitable well-known kind. Such solid support matrix for coupling a novel protein or polypeptide of the present invention might comprise, e.g., one of the following, without being limited thereto: resins, membranes, filters, columns, capillaries, particles, monoliths, fibers, pads, gels, slides, plates, cassettes, or any other format commonly used in chromatography and known to someone skilled in the art.

[0137] In one embodiment, the matrix is comprised of substantially spherical beads, for example agarose beads (for example, a polysaccharide polymer material in crosslinked form also known as Sepharose). Matrices in particle form can be used as a packed bed or in a suspended form including expanded beds. In other embodiments, the solid support matrix is a membrane, for example a hydrogel membrane. In some embodiments, the affinity purification can involve a membrane as a matrix to which the binding ligand for dsRNA is covalently bound. The solid support can also be in the form of a membrane in a cartridge.

[0138] In various embodiments, the affinity purification involves a chromatography column containing a solid support matrix to which a novel binding ligand for dsRNA of the present invention is covalently bound. The binding ligand for dsRNA as described above, may be attached to a suitable solid support matrix via conventional coupling techniques. Methods for immobilization of protein ligands to solid supports are well-known in the field of protein engineering and purification and can easily be performed by a skilled person in the art using standard techniques and equipment.

[0139] In various aspects and / or embodiments of the present invention, the binding ligand for dsRNA as disclosed herein including novel binding ligands for dsRNA generated or obtained by any methods as described herein are conjugated to a solid support. In some embodiments, the dsRNA binding ligand comprises an attachment site for site-specific covalent coupling of the dsRNA binding ligand to a solid support. Specific attachment sites comprise, but are not limited to, natural amino acids, such as cysteine or lysine, which enable specific chemical reactions with a reactive group of the solid phase, or a linker between the solid phase and the protein. In some embodiments, the fusion protein comprises the Protein A-like protein with attachment site is N-terminal of the dsRNA binding ligand. In preferred embodiments, the fusion protein comprising from N- to C-terminal orientation the Protein A-like protein as described herein and at least one dsRNA binding ligand as described herein wherein the Protein A-like protein has a cysteine in the region of amino acids 10-20, preferably in position 12 fo SEQ ID NOs: 20, 31 , or 33, or an amino acid sequence with at least 85 % sequence identity to the sequence of SEQ ID NOs: 20, 31 , or 33, including any of at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20, 31 , or 33.

[0140] In some embodiments, the binding ligand for dsRNA can also comprise additional amino acid residues at the N- and / or C-terminal end, such as for example an additional sequence at the N- and / or C-terminal end. Additional sequences may include for example sequences at introduced, e.g., for purification or detection. Typical examples for such sequences include, but are not limited to, Strep-tags, oligohistidine-tags, glutathione S-transferase, maltose- binding protein, inteins, intein fragments, or albumin-binding domain of protein G, or others. In other embodiments, additional amino acid sequences can include one or more peptide sequences that confer an affinity to certain chromatography column materials. The binding ligand for dsRNA may include specific attachment sites for the attachment to solid supports. In some embodiments, the specific attachment site comprises cysteine or lysine at the C- terminal end. In preferred embodiments, the fusion protein comprises cysteine as specific attachment site at the N-terminal end.

[0141] In another aspect, use of the affinity separation matrix according to the invention in affinity purification is provided. In preferred embodiments, use of the affinity separation matrix in the affinity purification of a ribonucleic acid preparation is provided.

[0142] In a further aspect, a kit for use in a method of purifying a ribonucleic acid preparation comprising the affinity separation matrix or the fusion protein as described herein is provided.

[0143] In various embodiments, the ribonucleic acid preparation is a mRNA preparation in the affinity separation matrix according to the invention, or the method as described herein, or the use as described herein, or the kit according to the invention. In preferred embodiments, mRNA is therapeutic mRNA.

[0144] In various embodiments, the ribonucleic acid preparation is a ssRNA preparation, in the affinity separation matrix as described herein, or the method as described herein, or the use as described herein, or the kit according to the invention. In preferred embodiments, ssRNA is inhibitory ssRNA, more preferably wherein the inhibitory ssRNA is therapeutic inhibitory ssRNA.

[0145] As described herein, a ..therapeutic inhibitory ssRNA" refers to ..inhibitory ssRNA for use in therapy". As described herein, ..therapy" encompasses the therapeutic treatment of diseases or disorders that benefit from, or require, targeting and regulating gene expression (inside target cells).

[0146] In another aspect, a method of production of a ssRNA preparation is provided by capturing the contaminant dsRNA, without non-specific degradation of the intended ssRNA, e.g., mRNA product. Said method of production of a ssRNA preparation comprises the following steps:

[0147] (i) providing a preparation that contains a dsRNA and ssRNA;

[0148] (ii) providing the affinity separation matrix as described herein;

[0149] (iii) contacting the preparation of (i) with the affinity separation matrix of (ii) under conditions that allow capturing of dsRNA by the dsRNA binding ligand;

[0150] (iv) separating the complex of the dsRNA bound to the affinity separation matrix from said preparation; and

[0151] (v) obtaining the ssRNA preparation.

[0152] Method of quantification of dsRNA. Methods to determine the presence of dsRNA in liquid samples can be quantitative or qualitative. Such methods are well-known to the person skilled in the art and can be selected, for instance without limitation, from the following methods that are well established in the art: enzyme-linked immunosorbent assay (ELISA), enzymatic reactions, surface plasmon resonance (SPR), Bio-layer interferometry (BLI) or chromatography.

[0153] Polynucleotides, vectors, host cells. One embodiment covers an isolated polynucleotide or nucleic acid molecule encoding a binding ligand for dsRNA comprising at least one dsRNA binding domain as described herein. A further embodiment also encompasses proteins encoded by said polynucleotides.

[0154] Further provided is a vector, in particular an expression vector, comprising the isolated polynucleotide or nucleic acid molecule for the binding ligand for dsRNA comprising at least one dsRNA binding domain as described herein, as well as a host cell comprising the isolated polynucleotide or the expression vector. For example, one or more polynucleotides, which encode the binding ligand for dsRNA comprising at least one dsRNA binding domain as described herein may be expressed in a suitable host and the produced binding ligand may be isolated. A vector means any molecule or entity, such as, without limitation, nucleic acid, plasmid, bacteriophage or virus, that can be used for transfer of protein-encoding information into a host cell. Suitable vectors that may be applied are known in the art. Furthermore, an isolated cell comprising a polynucleotide or nucleic acid, or a vector as disclosed herein is provided. Suitable host cells include prokaryotes or eukaryotes, or example a microbial host cell, including without limitation a bacterial host cell and a yeast host cell, or a non-human host cell carrying a vector. Suitable bacterial expression host cells or systems are well-known in the art. Various mammalian or insect cell culture systems are known in the art and can also be applied to express recombinant proteins.

[0155] The following Examples are provided for further illustration of the invention. The invention, however is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above description.

[0156] EXAMPLES

[0157] The following Examples are provided for further illustration of the invention. The invention, however, is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above specification and preferred embodiments of the invention. For a complete disclosure of the invention, reference is made also to the literature, which is cited throughout the specification and which is incorporated herein by reference in its entirety. Those examples that are not covered by the appending claims are given for comparative purposes only.

[0158] Example 1 : Soluble expression and purification of dsRNA binding domains and ligands of the invention

[0159] The untagged proteins were purified by using AKTA FPLC systems (Cytiva). Initial small-scale purifications were performed via StrepTactin XT (I BA Lifesciences) followed by size exclusion chromatography (Cytiva: Superdex200 26 / 600 or Sephacryl S200) in 20 mM citric acid, 150 mM NaCI, 1 mM EDTA, 5mM TCEP pH 6.5. For the scaled-up process, the initial capturing step was performed by using a SP65 cation exchange chromatography column (Purolite; binding buffer: 50 mM acetate, 1mM EDTA pH 4,0; wash bufferl : 50 mM acetate, 1M NaCI, 1mM EDTA pH 4,0; wash buffer2: 50 mM acetate, 1 mM EDTA pH 5,0; elution buffer: 50 mM Tris, 100 mM NaCI, 1 mM DTT, 1 mM EDTA pH 9,5). Afterwards the pooled elution fractions were polished using an anion exchange chromatography (Q65, Purolite; binding buffer: 50 mM sodium hydrogen carbonate, 1 mM DTT, 1 mM EDTA pH 9,0; elution buffer: 50 mM sodium hydrogen carbonate, 1 mM DTT, 1M NaCI, 1 mM EDTA pH 9,0). Finally, the protein was buffer exchanged and concentrated to concentrations >40 mg / mL using a SartoFlow Smart TFF system (Sartorius). Protein concentrations were determined by absorbance measurement at 280 nm using the molar absorption coefficient. Further analysis included Reversed phase chromatography (RP-HPLC) and size exclusion chromatography (SE-HPLC). RP-HPLC has been performed using an Agilent 1290 Infinity II System and a PLRP-S (5 pm, 300 A) column (Agilent). The resulting purity of all proteins was >95 %. SE-HPLC has been performed using an Agilent 1290 Infinity II System and a Superdex75 increase 5 / 150 GL or Superdex200 increase 5 / 150 GL (both Cytiva). No aggregation or oligomers were obtained.

[0160] Example 2: Binding affinity to dsRNA and to ssRNA (Binding analysis by BLI)

[0161] High-Throughput micro-scale purified proteins or lab scale purified proteins were characterized for binding against dsRNA and ssRNA using an ForteBio - Octet QK384 blue BLI system. Biotinylated ssRNA and dsRNA of different length (20, 40 & 200 nucleotides per strand) were immobilized on equilibrated Octet SA Biosensors (Sartorius) in Octetbuffer (50mM Tris, 150mM NaCI, 0.02% Tween, pH 7.3 RNase-free) at 1000 rpm for 600 s. After a wash step with Octetbuffer, the binding assay was started. Sensors underwent the following steps at room temperature at 1000 rpm: Baseline / wash (Octetbuffer; 60 s), association (ligand at 30-1000 nM in Octetbuffer; 120 s), dissociation (Octetbuffer; 120 s), regeneration (4 M Guanidine- HCI / Octetbuffer; 3x5 s in alternation). Data evaluation was performed with the instrument’s software using a Langmuir 1 :1 model. Evaluated dissociation constants (KD) were standardized against the immobilized protein and indicated. The assay is based on phase shift correlation generated between interference patterns of sensor surfaces. Determined dissociation constants for 40 bp dsRNA were below 200 nM, determined dissociation constants for 200 bp dsRNA were below 50 nM (see Table 1).

[0162] The fusion proteins comprise a non-dsRNA binding moiety of SEQ ID NO: 33 or SEQ ID NO: 31 at the N-terminus. SEQ ID NO: 31 was tested via BLI analysis and showed no detectable binding to dsRNA. SEQ ID NO: 31 has two additional amino acids I and A in position 57 and 58, compared to SEQ ID NO: 33, that should have no effect on the binding, thus it is expected that SEQ ID NO: 33 shows no detectable binding to dsRNA. Some of the ligands tested had a Strep-Tag at the C-terminus in these analyses.

[0163] Table 1. Binding affinity of ligands to dsRNA and ssRNA

[0164] Example 3: Affinity chromatography

[0165] High coupling density to resin. Affinity ligands were purified to homogeneity and immobilized at 20 mg per mL on activated Praesto Epoxy Jetted 50 (Purolite) according to the manufacturer’s instructions, coupling conditions: 45 °C for 3 h, pH 9.5. The achieved coupling density was about 15-20 mg / ml for coupled ligands.

[0166] DBC10%. Coupled resin was packed into Omnifit 5 / 100 column (Diba). 40 nucleotides per strand dsRNA was used as load (cone. 43 pg / ml; in binding buffer 50 mM Tris, 150 mM NaCI, pH 7.3). The sample was applied to the matrix comprising immobilized affinity ligand until 10 % target breakthrough at 6 min residence time. Unbound sample was washed off with binding buffer. The loaded sample was quantified and calculated as dynamic binding capacity DBC10 %. The binding capacity (DBC10) for affinity ligand 232075 was 3,2 mg / ml, for 232077 3,6 mg / ml, for 2320804,1 mg / ml. Bound dsRNA was eluted with 100 mM citric acid, 5 M NaCI pH 2.0.

[0167] Caustic stability. Caustic stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h. Columns were incubated with 0.1 M NaOH for 10 h at room temperature (22 °C + / - 3 °C); 1 set was measured. DBC10 was measured after incubation with 0.1 M NaOH and compared to DBC10 values without NaOH incubation. After 10 h in 0.1 M NaOH, 232075 revealed > 28 %, 232077 had > 58 %, and 232080 had 10 % remaining target binding capacity compared to initial value (DBC10 in %) (see Table 2).

[0168] Acidic stability. Acidic stability reflects the remaining binding capacity (in %) compared to the binding capacity at 0 h. Columns with immobilized ligand 232077 were incubated with 1 M acetic acid for 5 h at room temperature (22 °C + / - 3 °C); one set was measured. Initial DBC10 value was 3.42 mg per ml resin and after static incubation for 5 h in 1 M acetic acid the DBC10 value was unchanged and therefor remaining capacity was 100 %.

[0169] Table 2. Caustic stability of selected dsRNA binding ligands

[0170] Example 4: Affinity chromatography (232077, 232080, 232075)

[0171] AIC chromatography. Coupled resin was packed into Omnifit 6.6 / 100 column (Diba). Resin with coupled affinity ligand 232075 (SEQ ID NO: 30), 232077 (SEQ ID NO: 21), or 232080 (SEQ ID NO: 27) was loaded with 500 l sample containing 500 pg mRNA with varying dsRNA contamination (1.55 - 0.22 % dsRNA, loading buffer 50 mM Tris, NaCI between 550 mM and 650 mM). For initial measurements, a loading buffer with 50 mM Tris, 150 mM NaCI, pH 7.3 was used. A gradient was used from 0-100 % elution buffer (50 mM Tris, 1 M NaCI pH 7.3) for ssRNA elution. ssRNA elutes within this gradient from the resin with coupled affinity ligand, whereas dsRNA elutes only at harsher conditions (100 mM citric acid, 5 M NaCI pH 2.0). To remove dsRNA from target mRNA, a flow-through mode (FT mode) is applied. To ensure high yield (recovery) of ssRNA in flow through and optimized LRV (dsRNA / mRNA), salt concentrations were for resins with coupled ligands 232075 or 232077 550 mM NaCI and for the resin with coupled ligand 232080 650 mM NaCI.

[0172] Depletion of dsRNA (LRV) analysed via Elisa. The reduction of dsRNA in the flow through was analysed. Low dsRNA contamination was not determinable by 260 nm. An ELISA assay was conducted wherein the dsRNA was immobilized between two specific antibodies (mouse monoclonal anti-dsRNA lgG2a clone K1 (Jena Bioscience, RNT-SCI- 10020500) and mouse monoclonal anti-dsRNA lgG2a clone J2 (Jena Bioscience, RNT-SCI-10010500) coupled to a horse horseradish peroxidase (HRP). For all dsRNA ligands, under this condition a log reduction value (LRV) for dsRNA contamination between 0.22 % and 1.58 % of at least 1.4 was achieved (see Table 3).

[0173] Table 3. Depletion of dsRNA

[0174] Example 5: Recovery of ssRNA

[0175] To analyze the amount of ssRNA in the flow through (recovery) 5 ml ssRNA sample (1.9 mg ssRNA) with a contamination of 0.041% dsRNA was used, in order to reduce systematic error, which occur by loading small volumes onto column due to void volumes of device. For affinity chromatography, 0.4-0.5 ml resin with immobilized ligand (232075, 232077, 232080) was used, packed into Omnfit 6.6 / 100 (DEBA) column. Samples were injected with salt concentration of 550 mM NaCI (232075, 232077) or 650 nM NaCI (232080) in loading buffer to ensure FT-Mode for ssRNA. The recovery was obtained by measuring the absorption at 260 nm. Depletion of dsRNA have been followed by an ELISA assay. For all ligands, the recovery was higher than 98 % and LRV was under used conditions higher than 1.2 (see Table 4).

[0176] Table 4. Recovery of ssRNA in FT-Mode

[0177] Table 5. Amino acid sequences KNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAA EKALRSFVFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYV GSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPNYIGLINRIAQKKRLTVNYE QCASGVHGPEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILS (SEQ ID NO: 23; fusion protein 231892; comprising dsRNA binding ligand comprising SEQ ID NOs: 1 and 6, linker SEQ ID NO: 8, and non dsRNA binding protein SEQ ID NO: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKLP KNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAA EKALRSFVEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQCASGVHG PEGFHYKCKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILS (SEQ ID NO: 24; fusion protein 231893; comprising dsRNA binding ligand comprising SEQ ID NOs: 1 and 6, linker SEQ ID NO: 9, and non dsRNA binding protein SEQ ID NO: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKGF FMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKN AAAKLAVEI LN KEKKAVSPLLLTTTNSSEGLSMGPSGKN PVM I LN ELRPGLKYDFLSE SGESHAKSFVMSWVDGQFFEGSGRNKKLAKARAAQSALAAIFN (SEQ ID NO: 25; fusion protein 231894; comprising dsRNA binding ligand comprising SEQ ID NOs: 5 and 2, linker SEQ ID NO: 9, and non dsRNA binding protein SEQ ID NO: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKGF FMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKN AAAKLAVEILNKFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPP FYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPG LKYDFLSESGESHAKSFVMSWVDGQFFEGSGRNKKLAKARAAQSALAAIFN (SEQ ID NO: 26; fusion protein 231895; comprising dsRNA binding ligand comprising SEQ IDs: 5 and 2, linker SEQ ID: 8, and non dsRNA binding protein SEQ ID: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKGF FMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKN AAAKLAVEILNKPTGAEGRDSSKGEDSAEETEAKPAWAPAPVVEAVSTPSAAFPSD ATAEQGPI LTKHGKNPVM ELN EKRRGLKYELISETGGSH DKRFVM EVEVDGQKFQG AGSNKKVAKAYAALAALEKLFPSAWSHPQFEK (SEQ ID NO: 27; fusion protein 232080; comprising dsRNA binding ligand comprising SEQ ID NOs: 5 and 4, linker SEQ ID NO: 7, and non dsRNA binding protein SEQ ID NO: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKGF FMEELNTYRQKQGWLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKN AAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGHGKNPVMELNEKRRGLKYELISET GGSH DKRFVM EVEVDGQKFQGAGSN KKVAKAYAALAALEKLFPSAWSH PQFEK (SEQ ID NO: 28; fusion protein 231896; comprising dsRNA binding ligand comprising SEQ IDs: 5 and 4, linker SEQ ID: 9, and non dsRNA binding protein SEQ ID: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKEP PQAMNALMRLNQLKPGLQYKLVSQTGPVHAPIFTMSVEVDGNSFEASGPSKKTAKL HVAVKVLQDMGLFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEP PFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRP

[0178] GLKYDFLSESGESHAKSFVMSVWDGQFFEGSGRNKKLAKARAAQSALAAIFN (SEQ ID NO: 29; fusion protein 231898; comprising dsRNA binding ligand comprising SEQ IDs: 3 and 2, linker SEQ ID: 8, and non dsRNA binding protein SEQ ID: 33) AQHDKIQQAADCEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKA

[0179] QHDKIQQAADKEILHLPNLTEEQRNKFRQSLRDDPSVSAEILAEAKKLNDAQAPKEP

[0180] PQAMNALMRLNQLKPGLQYKLVSQTGPVHAPIFTMSVEVDGNSFEASGPSKKTAKL

[0181] HVAVKVLQDMGLPTGAEGRDSSKGEDSAEETEAKPAVVAPAPVVEAVSTPSAAFPS

Claims

CLAIMS1. An affinity separation matrix for purifying a ribonucleic acid preparation comprising a binding ligand for double stranded RNA (dsRNA), wherein the dsRNA binding ligand comprises at least one dsRNA binding domain comprising an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 90 % identity thereto.

2. The affinity separation matrix according to claim 1 , wherein the dsRNA binding ligand comprises the dsRNA binding domains comprising: a) the amino acid sequences of SEQ ID NOs: 1 and 4, or sequences with at least 90 % identity thereto; b) the amino acid sequences of SEQ ID NOs: 4 and 5, or sequences with at least 90 % identity thereto; or c) the amino acid sequences of SEQ ID NOs: 3 and 4, or sequences with at least 90 % identity thereto.

3. The affinity separation matrix according to claim 1 or 2, wherein the dsRNA binding ligand has more than 10-fold binding affinity for dsRNA as compared to the binding affinity for ssRNA under the same conditions, preferably wherein the dsRNA binding ligand has more than 10-fold binding affinity for about 40 bp dsRNA as compared to the binding affinity for about 40 nt ssRNA under the same conditions.

4. The affinity separation matrix of any one of claims 1 to 3, wherein the dsRNA binding ligand has a binding affinity for dsRNA of about 200 nM or less, in particular wherein the dsRNA binding ligand has a binding affinity of about 200 nM or less for dsRNA with a length of about 40 bp.

5. The affinity separation matrix of any one of claims 1 to 4, wherein the dsRNA binding ligand comprises at least two dsRNA binding domains, which are linked by a linker, preferably wherein the linker is a peptide linker, in particular wherein the peptide linker has a length of at least 5 amino acid residues.

6. The affinity separation matrix according to claim 5, wherein the peptide linker comprises the amino acid sequence of any one of SEQ I D NOs: 7 to 9, or an amino acid sequence with at least 90 % identity thereto.

7. The affinity separation matrix according to any one of claims 1 to 6, wherein the dsRNA binding ligand is fused to a Protein A domain or a Protein A-like protein, preferablywherein the Protein A domain or the Protein A-like protein lacks detectable binding to dsRNA.

8. A fusion protein comprising the dsRNA binding ligand according to any one of claims 1 to 6 and a Protein A domain or Protein A-like protein, preferably wherein the Protein A- like protein lacks detectable binding affinity to dsRNA.

9. The fusion protein according to claim 8, wherein the fusion protein comprises an amino acid sequence with at least 90 % amino acid sequence identity to any one of SEQ ID NOs: 21-30.

10. A method of purifying a ribonucleic acid preparation, comprising: a) providing a ribonucleic acid preparation containing single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), b) contacting the ribonucleic acid preparation of a) with the affinity separation matrix of any one of claims 1 to 7, or the fusion protein of claim 8 or 9, to capture the dsRNA, c) separating the ssRNA from the captured dsRNA.11 . Use of the affinity separation matrix of any one of claims 1 to 7, or the fusion protein of claim 8 or 9, in affinity purification, in particular in affinity purification of a ribonucleic acid preparation.

12. A kit for use in a method of purifying a ribonucleic acid preparation comprising the affinity separation matrix of any one of claims 1 to 7, or the fusion protein of claim 8 or 9.

13. The affinity separation matrix of any one of claims 1 to 7, or the method of claim 10, or the use of claim 11 , or the kit of claim 12, wherein the ribonucleic acid preparation is a mRNA preparation or a ssRNA preparation, preferably wherein the mRNA is therapeutic mRNA or the ssRNA is inhibitory ssRNA, respectively, e.g., wherein the inhibitory ssRNA is therapeutic inhibitory ssRNA.

14. A method of production of a single-stranded RNA (ssRNA) preparation, the method comprising:(i) providing a preparation that contains double-stranded RNA (dsRNA) and ssRNA;(ii) providing the affinity separation matrix of any one of claims 1 to 7, or the fusion protein of claim 8 or 9;(iii) contacting the preparation of (i) with the affinity separation matrix or the fusion protein of (ii) under conditions that allow capturing of dsRNA by the dsRNA binding ligand;(iv) separating the complex of the dsRNA bound to the affinity separation matrix or the fusion protein from said preparation; and(v) obtaining the ssRNA preparation.

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