WO2025245419A2

WO2025245419A2 - Affinity agents for rna purification

Info

Publication number: WO2025245419A2
Application number: PCT/US2025/030712
Authority: WO
Inventors: Nathaniel Clark; Thomas Scanlon
Original assignee: Repligen Corp
Current assignee: Repligen Corp
Priority date: 2024-05-23
Filing date: 2025-05-23
Publication date: 2025-11-27
Anticipated expiration: 2026-11-23

Abstract

Provided are compositions, systems and methods for removal of double stranded RNA from a mixture utilizing affinity agents having high binding affinity and selectivity for double stranded RNA over single stranded RNA.

Description

AFFINITY AGENTS FOR RNA PURIFICATION

REFERENCE TO A SEQUENCE LISTING

[0001] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML sequence listing, created on May 21, 2025, is named 158000225WO_Sequence Listing and is 26,863 bytes in size.

BACKGROUND

[0002] Double stranded RNA (dsRNA) is a common impurity created during RNA manufacturing. Contaminating dsRNA fragments are difficult to separate from single-stranded RNA (ssRNA) product. However, dsRNA is highly immunogenic and if not removed can lead to inflammation, reduced efficacy, and increased toxicity of therapeutic RNA molecules.

Accordingly, there exists a need to selectively remove dsRNA impurities from ssRNA product during RNA manufacturing.

BRIEF SUMMARY

[0003] The present invention provides compositions, systems and methods for removal of dsRNA from a mixture containing ssRNA. Provided are affinity ligands having high selectivity for dsRNA over ssRNA. Also provided are resins functionalized with an affinity ligand as described herein. Related systems and methods for RNA manufacturing are also provided.

[0004] In one aspect, provided is an affinity ligand of Formula I, or a multimer thereof Li-Di- L2-DS-L3-DT-L4 (Formula I) where Li, L2, L3, and L4 are each independently absent or a linker, Di is a dsRNA-binding domain includes an amino acid sequence 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing, Ds is an optional structural domain, and DT is an optional C-terminal tag domain.

[0005] In one aspect, provided is an affinity chromatography matrix comprising a solid support and a double stranded RNA (dsRNA)-binding polypeptide ligand covalently attached to the solid support, where the ligand includes from one to six dsRNA-binding domains, where each dsRNA-binding domain is defined by a core sequence of from 60-200 amino acids, optionally from 70-200 amino acids, and where the core sequence is the same or different in each of the from one to six dsRNA-binding domains.

[0006] The affinity chromatography matrix may also include where each dsRNA-binding domain includes at a C-terminal end of its core sequence one or more of a spacer molecule, a reactive thiol- or nitrogen-containing amino acid, a structural domain, or a peptide, polypeptide, or protein tag. The affinity chromatography matrix may also include where the C- terminal spacer molecule is selected from an amino acid, a polypeptide of from 1-60 amino acids, or a non-polypeptide molecule, the C-terminal reactive thiol- or nitrogen-containing amino acid is cysteine or lysine, and the C-terminal tag is a cysteine. The affinity chromatography matrix may also include where the core sequence includes a 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing.

[0007] The affinity chromatography matrix may also include where the ligand is a polypeptide of Formula I, or a multimer thereof L1-D1-L2-DS-L3-DT-L4 (Formula I) where Li, L2, L3, and L4 are each independently absent or a linker, Di is a dsRNA-binding domain includes a 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing, Ds is an optional structural domain, and DT is an optional C-terminal tag domain.

[0008] In aspects of any of the foregoing, the affinity ligand may include from two to four dsRNA-binding domains, optionally where the ligand is a homomultimer or a heteromultimer with respect to the dsRNA-binding domains. In aspects, the ligand consists of SEQ ID NO: 20, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the ligand consists of SEQ ID NO: 21, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the ligand consists of SEQ ID NO: 24, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the ligand consists of an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the ligand consists of an amino acid sequence of SEQ ID NO: 26, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0009] In accordance with aspects of any of the foregoing, the affinity chromatography matrix includes a solid support with the affinity ligand covalently attached thereto. The affinity chromatography matrix may also include where the solid support comprises the ligand at a density of from about 10-20 mg/mL, or about 15-20 mg/ml. In aspects, the affinity matrix has a dynamic binding capacity of at least about 5 mg/ml for dsRNA. In aspects, the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith. In aspects, the discrete polymeric particles are particles of a polysaccharide or synthetic polymer, optionally where the polysaccharide is agarose or cellulose, or a stabilized variant or derivative thereof, and the synthetic polymer is a polymethacrylate polymer.

[0010] Also provided are polynucleotides encoding the dsRNA-binding polypeptide of any one of SEQ ID NOs 1-26 and recombinant cells or viral particles comprising a polynucleotide encoding the dsRNA-binding polypeptide of any one of SEQ ID NOs 1-26.

[0011] Also provided are methods for depleting dsRNA from an aqueous mixture comprising dsRNA and ssRNA, the methods including contacting the mixture with an affinity chromatography matrix functionalized with dsRNA-binding ligand, as described herein, under conditions suitable for binding of the dsRNA to the ligand, and collecting the ssRNA in the flow-through from the affinity matrix. The method may also include where the contacting occurs as the mixture flows through a chromatography column comprising an affinity chromatography matrix as described herein. The method may also include buffering the mixture comprising dsRNA and ssRNA to a pH of from 7.2 to 10 and adjusting a salt concentration of the mixture to from about 0.3 to 2.5 M sodium chloride (NaCl) prior to contacting the mixture with the affinity chromatography matrix. The method may also include an additional step of eluting the dsRNA from the affinity chromatography matrix following the step of collecting the ssRNA. The method may also include a cleaning in place step following elution of the dsRNA from the affinity chromatography matrix, comprising contacting the affinity matrix with an alkaline solution comprising from 0.01-0.05 M NaOH or 6 M guanidine hydrochloride for a period of at least 10 hours, optionally at least 20 hours or at least 30 hours. [0012] Also provided are systems for RNA manufacture comprising an in vitro transcription unit, an RNA purification unit, and a ssRNA purification unit, where the ssRNA purification unit includes an affinity chromatography apparatus comprising an affinity matrix functionalized with a dsRNA-binding ligand as described herein. The system may also include where the RNA purification unit comprises an RNA affinity column, optionally where the RNA affinity column is an oligo(dT)-cellulose column or a poly(U)-Sepharose column. The system may also include where the ssRNA purification unit is placed before or after the RNA affinity column. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 is an image of a dot blot showing salt and pH conditions for removal of dsRNA from in vitro transcribed firefly luciferase ssRNA.

[0014] FIG. 2 is an image of an immuno-northern blot of dsRNA depleted firefly luciferase samples (top panel). The bottom panel shows RNA loaded into each lane, visualized using a SYBR safe nucleic acid stain.

[0015] FIG. 3 shows a chromatographic trace of binding capacity (top panel) for a synthetic 500 base-pair dsRNA molecule and an immuno-northern blot of the post-column fractions (bottom panel).

[0016] FIG. 4 is a bar graph quantitating the selectivity for dsRNA over ssRNA as a ratio of bound dsRNA: ssRNA for affinity matrices functionalized with different dsRNA-binding affinity ligands. The numbers below each bar correspond to the sequence identifier for the affinity ligand. Each bar represents the ratio of bound dsRNA to ssRNA. Bars labeled 1-10 in the figure represent resins prepared with the affinity ligands of Formula I, as described infra.

[0017] FIG. 5 is a bar graph showing percentage double-stranded RNA (% dsRNA) bound by resins functionalized with either (i) a prior art Thermotoga maritima RNAse III based ligand (SeqX), or a ds-RNA binding ligand of the invention comprising a (ii) monomer (SEQ ID NO: 17) or (iii) tetramer (SEQ ID NO: 18), of a dsRNA-binding domain defined by SEQ ID NO: 6. A no resin condition is used as a negative control. A reference cellulose-based method for removal of dsRNA, described in Baiersdbrfer et al., Mol Ther Nucleic Acids 2019 Feb 27;15:26-35, is used as a positive control (cellulose/ethanol).

DETAILED DESCRIPTION

[0018] Provided are affinity ligands, which may be referred to herein as “affinity agents” or in some instances simply as “ligands”, and related compositions, including affinity resins functionalized with the ligands, and related methods, including methods of isolating a target molecule utilizing the affinity ligands and resins described herein. The term “affinity ligand” refers to a molecule that binds with high affinity to a target molecule, typically with a dissociation constant, ko, in the nanomolar range. Affinity resins prepared with the ligands disclosed herein are generally useful in methods of detection, isolation, and/or purification of the target molecule. The affinity ligands described herein may be characterized as polypeptides or proteins and the terms “polypeptide” and “protein” are used interchangeably herein. In the context of the present invention, the target molecule is double stranded RNA (dsRNA). The target molecule, dsRNA, is a significant contaminant produced as a transcriptional by-product during in vitro transcription of messenger RNA (mRNA). Accordingly, affinity resins functionalized with the ligands described here are useful in removing contaminating dsRNA from mixtures of single-stranded (ssRNA), including mRNA ribosomal RNA (rRNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), microRNA (miRNA), short-hairpin RNA (shRNA), and non-coding RNA (ncRNA), or other forms of ssRNA obtained by in vitro transcription.

[0019] Accordingly, provided are double stranded RNA (dsRNA)-binding polypeptides, which may also be referred to herein as dsRNA-binding ligands, chromatography matrices functionalized with the dsRNA-binding polypeptides, and related methods for separating dsRNA from ssRNA, as well as related systems and methods of RNA manufacture, including mRNA.

[0020] The dsRNA-binding ligands described herein comprise one or more RNA-binding domains. In aspects, the one or more RNA-binding domains is a domain of a bacterial, viral, or human protein, or a dsRNA-binding polypeptide or derivative of such RNA-binding domain. In aspects, the one or more RNA-binding domains is a domain of a bacterial protein, or a dsRNA- binding polypeptide or derivative of such RNA-binding domain, where the bacterial protein is an RNAase enzyme or an RNA polymerase binding protein. In aspects, the one or more RNA- binding domains is a domain of a viral protein, or a dsRNA-binding polypeptide or derivative of such RNA-binding domain, where the viral protein is a viral RNA binding protein. In aspects, the one or more RNA-binding domains is a domain of a human protein, or a dsRNA- binding polypeptide or derivative of such RNA-binding domain, where the human protein is a Staufen family protein.

[0021] In aspects, provided is a dsRNA-binding ligand comprising two or more units of a dsRNA-binding domain comprising an amino acid sequence 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, SEQ ID NO: 6 and SEQ ID NO: 7, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity to any of the foregoing. An affinity ligand comprising two or more units of a dsRNA binding domain may be referred to as a “multimer” or by the number of units of the binding domain(s) present in the protein. For example an affinity ligand having two units of a binding domain may be referred to as a dimer. Similarly, an affinity ligand having three, four, five, or six units of a binding domain may be referred to as a trimer, a tetramer, a pentamer, or a hexamer, respectively. In some aspects, a multimer may comprise two or more units of the same binding domain. In some aspects, a multimer may comprise two or more units of different binding domains. In the context of a multimer, the two or more units of a binding domain are covalently attached to each other, either directly or via a linker molecule. Linker molecules are described in more detail infra, but generally a linker molecule is a single amino acid residue, a polypeptide, or a nonpolypeptide molecule.

[0022] In aspects, provided is an affinity ligand of Formula I:

L1-D1-L2-DS-L3-DT-L4 (Formula I) wherein

Li, L2, L3, and L4 are each independently absent or a linker;

Di is dsRNA-binding domain comprising an amino acid sequence 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, SEQ ID NO: 6 and SEQ ID NO: 7, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity to any of the foregoing, or a multimer thereof; Ds is an optional structural domain; and

DT is an optional C-terminal tag domain.

[0023] In aspects of Formula I, each of Li, L2, L3, and/or L4, if present, is a linker. In aspects, the linker is a peptide linker of from 1-12 amino acids in length, optionally from 1-10 amino acids in length or from 1-6 amino acids in length. In aspects, any one or more of Li, L2, L3, and/or L4, if present, is independently an alanine, a poly(glycine) peptide, poly(alanine) peptide, poly((glycine)(alanine)) peptide, or a poly((glycine)(serine)) peptide of from 1-5 amino acid residues in length. In other aspects, the linker may be as described in infra.

[0024] In aspects of Formula I, L4 is a C-terminal reactive thiol- or nitrogen-containing amino acid. In aspects, L4 is a C-terminal lysine or cysteine residue.

[0025] In aspects of Formula I, Ds is an optional non-target molecule binding structural domain. In aspects, the structural domain may enhance soluble protein expression and/or serve as a tag domain for use in affinity purification of the target molecule. Exemplary structural domains include glutathione S-transferase (GST), maltose binding protein (MBP), small ubiquitin-like modifier (SUMO), and Staphylococcal protein A (SpA). In aspects, Ds may comprise or consist of a polypeptide derivative of SpA that does not bind to the Fc region of immunoglobulin proteins. Additional exemplary structural domains include ecotin, the Z- domain of SpA, the albumin-binding domain of protein G, the cellulose-binding domain of endoglucanase, disulfide bond oxidoreductase, and Barnase, an enzymatically inactive variant an RNase enzyme of Bacillus amyloliquefaciens. See review by A. Malik, 3 Biotech. 2016 Feb 4;6(1):44.

[0026] In aspects of Formula I, the tag domain DT may include one or more C-terminal tags, as described in more detail infra. In aspects, DT comprises at least one affinity tag to facilitate isolation during manufacture and/or to facilitate detection of the affinity ligand or its target molecule.

[0027] In aspects of Formula I, Di is dsRNA-binding domain comprising a multimer of an amino acid sequence 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, SEQ ID NO: 6 and SEQ ID NO: 7, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity to any of the foregoing. In aspects, the multimer is a dimer, trimer, tetramer, pentamer, or hexamer. [0028] In aspects of Formula I, Di is a dsRNA-binding domain comprising an amino acid sequence as defined by SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer thereof. In aspects, the multimer is a dimer or trimer of an amino acid sequence defined by SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity thereto.

[0029] In aspects, a dsRNA-binding ligand described herein consists of a dsRBD amino acid sequence defined by SEQ ID NO: 21, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0030] In aspects, a dsRNA-binding ligand described herein consists of a Aa-dsRBD amino acid sequence defined by SEQ ID NO: 20, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0031] In aspects, a dsRNA-binding polypeptide described herein comprises a dsRNA-binding domain of a bacterial RNAse enzyme or a dsRNA-binding polypeptide or a derivative thereof. In aspects, the bacterial RNAse enzyme is an Aquifex aeolicus RNAselll enzyme. In aspects, the dsRNA-binding polypeptide comprises an amino acid sequence defined by SEQ ID NO: 6, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide consists of an amino acid sequence defined by SEQ ID NO: 20, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0032] In aspects, a dsRNA-binding polypeptide described herein comprises a dsRNA-binding domain of a bacterial RNA polymerase-binding protein or a dsRNA-binding polypeptide or derivative thereof. In aspects, the bacterial RNA polymerase-binding protein is a Mycobacterium tuberculosis RbpA protein. In aspects, the dsRNA-binding polypeptide comprises an amino acid sequence defined by SEQ ID NO: 2, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide consists of an amino acid sequence defined by SEQ ID NO: 25, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. [0033] In aspects, a dsRNA-binding polypeptide described herein comprises an RNA-binding viral protein or dsRNA-binding domain thereof, or a dsRNA-binding polypeptide or derivative thereof. In aspects, the dsRNA-binding polypeptide comprises or consists of a flock house virus protein or dsRNA-binding domain thereof, or a dsRNA-binding polypeptide or derivative thereof. In aspects, the dsRNA-binding polypeptide comprises an amino acid sequence defined by SEQ ID NO: 1, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide consists of an amino acid sequence defined by SEQ ID NO: 24, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide comprises or consists of a Carnation Italian ring-spot virus (CIRV) pl9 RNA binding protein or dsRNA- binding domain thereof, or a dsRNA-binding polypeptide or derivative thereof. In aspects, the dsRNA-binding polypeptide comprises an amino acid sequence defined by SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide consists of an amino acid sequence defined by SEQ ID NO: 22 or SEQ ID NO: 23, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0034] In aspects, a dsRNA-binding polypeptide described herein comprises an RNA-binding human protein or dsRNA-binding domain thereof, or a dsRNA-binding polypeptide or derivative thereof. In aspects, the dsRNA-binding polypeptide comprises or consists of a Staufen family protein or dsRNA-binding domain thereof, or a dsRNA-binding polypeptide or derivative thereof. In aspects, the dsRNA-binding polypeptide comprises an amino acid sequence defined by SEQ ID NO: 7, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity. In aspects, the dsRNA-binding polypeptide consists of an amino acid sequence defined by SEQ ID NO: 26, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0035] Also provided are chimeric proteins comprising a plurality of dsRNA-binding polypeptides as described herein. In this context, the chimeric protein is itself a dsRNA-binding polypeptide comprising two or more RNA-binding domains, each defined by a core sequence of from 70 to 200 amino acids, wherein each core sequence of the chimeric protein is independently 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, SEQ ID NO: 6 and SEQ ID NO: 7. Thus, in aspects, the chimeric protein may comprise multiples of the same dsRNA-binding polypeptide, forming a homomultimer, or different dsRNA-binding polypeptides, forming a heteromultimer. In aspects, the chimeric protein comprises two, three, four or five RNA-binding domains, each defined by a core sequence independently 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, SEQ ID NO: 6 and SEQ ID NO: 7. In this context, the core sequences may be separated by a linker or spacer, as described herein.

[0036] A dsRNA-binding polypeptide or chimeric protein as described herein may be functionalized with one or more N- or C-terminal modifications to facilitate its purification during manufacture, to facilitate its covalent attachment to a solid support to form an affinity chromatography matrix or resin as described herein, and/or to improve its stability or expression. In this context, the term “functionalized” refers to a modification by covalent attachment. The one or more C-terminal modifications may include a spacer or linker molecule, a peptide, polypeptide, or protein tag, a structural domain, and combinations thereof. In aspects, the one or more C-terminal modifications may include, for example, a linker molecule, a C-terminal reactive thiol- or nitrogen-containing amino acid, or a peptide, polypeptide, or protein tag.

[0037] In the context of the present disclosure, the terms “spacer” and “linker” are used interchangeably. In aspects where a dsRNA-binding polypeptide or chimeric protein as described herein is functionalized with a linker, the linker may consist of a single amino acid residue, a polypeptide, or a non-polypeptide molecule. In aspects, the single amino acid spacer may be an alanine, glycine^ serine, proline, aspartic acid, glutamic acid, or any natural or nonnatural amino acid residue. In aspects, the single amino acid spacer is a glycine, alanine, or serine residue. In aspects, the polypeptide linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In aspects, the linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In aspects, the linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, serine, and/or alanine. In some aspects, the linker is selected from a poly(glycine) peptide, a poly(alanine) peptide, a poly((glycine)(alanine)) peptide and a poly((glycine)(serine)) peptide. Polypeptide linkers may be from about 1 to 50 amino acids, from about 1 to 20 amino acids, from about 1 to 15 amino acids, from about 1 to 10 amino acids, from about 1 to 5 amino acids, from about 2 to 20 amino acids, from about 2 to 15 amino acids, from about 2 to 10 amino acids, or from about 2 to 5 amino acids in length. In aspects, the poly(glycine) peptide, poly(alanine) peptide, poly((glycine)(alanine)) peptide or a poly((glycine)(serine)) peptide linker consists of from about 2 to 10 amino acids, or from 2 to 6 amino acids, or from 2 to 4 amino acids.

[0038] In other aspects, the spacer or linker may be a non-polypeptide molecule, for example a substituted or unsubstituted C2-C30 alkyl spacer or a polyethylene glycol (PEG) spacer. In aspects, the C2-C30 alkyl spacer is -NH-(CH2)n-C(O)-, where n is from 2 to 30. In aspects, the alkyl spacer is substituted by a lower alkyl, e.g., Ci-Ce alkyl, acyl, halogen, -CN, -NH2, or phenyl. In aspects, a PEG spacer has a molecular weight of from about 100 to 5000 kDa, or from about 100 to 500 kDa.

[0039] In aspects, a dsRNA-binding polypeptide or chimeric protein described herein may be functionalized with a peptide, polypeptide, or protein tag, for example a C-terminal affinity tag, to facilitate its isolation during manufacture and/or its detection. The term “tag” refers to a polypeptide covalently attached to another polypeptide or protein, generally to facilitate isolation and/or detection of the tagged polypeptide or protein. Such tags may be from about 4 to 500 amino acids in length. Tags allow for isolation, detection and/or localization of the tagged polypeptide or protein using methods such as affinity purification or immunodetection with an appropriate antibody combined with detection e.g., by Western analyses, ELISA assays, or immunostaining. Exemplary tags that may be utilized include the bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxy carrier protein (BCCP), polyhistidine (His-tag), polyaspartate (Asp-tag), polycysteine (Cys-tag), polyphenylalanine (Phe-tag), glutathione 5-transferase (GST), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein-chitin binding domain (intein-CBD), a streptavidin/biotin-based tag such as streptavadin, streptavadin-binding peptide (SBP) or Strep-tag, a tandem affinity purification (TAP) tag, such as Protein A of Staphylococcus aureus and calmodulin-binding peptide (CBP), or other TAP variants, a short epitope tag such as FLAG, human influenza hemagglutinin (HA), c-myc epitope, T7, or Glu-Glu, HSV epitope, and small fluorescent proteins such as the green fluorescent protein (GFP) and derivatives thereof. [0040] In aspects, a dsRNA-binding polypeptide or chimeric protein described here may include C-terminal tag. In aspects, a dsRNA-binding polypeptide described here may include a combination of tags and linkers, for example two or more of a polypeptide tag, an N-terminal linker, a C-terminal linker, and a structural domain.

[0041] Also provided are affinity chromatography matrices comprising a solid support functionalized with a dsRNA-binding polypeptide or chimeric protein as described herein. In this context, the term “functionalized” refers to a modification of the solid support by covalent attachment of a plurality of dsRNA-binding polypeptides.

[0042] The dsRNA-binding polypeptides may be covalently attached to the solid support via a reactive thiol- or nitrogen-containing amino acid. Alternatively or additionally, the dsRNA- binding polypeptides may be covalently attached to the solid support via reactive epoxy or aldehyde groups of the support itself. Accordingly, in aspects, the polypeptides are covalently attached to the solid support directly, via a reactive thiol- or nitrogen-containing C-terminal amino acid of the polypeptide, or indirectly, via a polypeptide linker. In aspects, a dsRNA- binding polypeptide as described herein may be modified to include a C-terminal reactive amino acid, e.g., a reactive thiol- or nitrogen-containing C-terminal amino acid, to facilitate covalent attachment of the polypeptide to the solid support. In aspects, the reactive amino acid is a lysine or cysteine. In aspects where the dsRNA-binding polypeptide is covalently attached to the solid support via a C-terminal polypeptide linker, the C-terminal polypeptide linker may comprise from 2 to 50 amino acids, from 2 to 25 amino acids, from 2 to 15 amino acids, from 2 to 10 amino acids, or from 2 to 5 amino acids. In aspects, the C-terminal polypeptide linker comprises one or more reactive amino acids e.g., a reactive thiol- or nitrogen-containing amino acid, to facilitate covalent attachment of the linker to the solid support. In aspects, the one or more reactive amino acids includes one or more lysine and/or cysteine residues.

[0043] In aspects, the dsRNA-binding polypeptides may be covalently attached to the solid support via a C-terminal polypeptide linker and a structural domain.

[0044] In aspects, the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith. The discrete polymeric particles may be made from a polysaccharide such as agar, agarose, dextran, starch, cellulose, pullulan, etc., and stabilized variants and derivatives thereof; or from a synthetic polymer such as polystyrene, polyvinylether, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, etc. In aspects, the discrete particles may have a volume- weighted median diameter (d50,v or dv50) in the range of about 20 to 100 micrometers, or 40 to 90 micrometers, or 45 to 55 micrometers, or 80 to 90 micrometers. In aspects, the dv50 of the particles is 50 or 85 microns. In aspects, the particles are made from a material selected from agarose or a stabilized derivative thereof such as Praesto®Pure or Praesto® letted A50, having a volume-weighted median diameter (d50,v) of about 50 or about 85 micrometers or having a d50v of from about 45 to 90 micrometers. In aspects, the particles are made from a material selected from agarose or a stabilized derivative thereof such as Sepharose™ or Superflow™ agarose. In aspects, the particles are discrete polymeric particles comprising a polymethacrylate polymer and having a d50v of from about 45 to 55 micrometers, or from about 47-50 micrometers.

[0045] In aspects, provided is an affinity chromatography matrix functionalized with at least about 1 to 20 mg/ml dsRNA-binding polypeptides or chimeric proteins as described herein, for example about 1 to 5 mg/ml or 5 to 15 mg/ml or 10-20 mg/ml or 15 to 20 mg/ml. In aspects, a column comprising the affinity chromatography matrix has a dynamic binding capacity (DBC) of at least about 0.1 to 0.5 g/L or 0.1 to 0.5 mg/ml for dsRNA. In aspects, a column comprising the affinity chromatography matrix has a dynamic binding capacity (DBC) of at least about 0.2 g/L or 0.2 mg/ml, 0.3 g/L or 0.3 mg/ml, 0.4 g/L or 0.4 mg/ml, or 0.5 g/L or 0.5 mg/ml for dsRNA.

[0046] Suitable solid supports include, for example, agarose and stabilized derivatives of agarose (e.g. Praesto®Pure, Praesto® Jetted A50, Sepharose 6B, Sepharose Fast Flow, etc.), cellulose or derivatives of cellulose, controlled pore glass, monolith (e.g. CIM® monoliths), silica, zirconium oxide (e.g. CM Zirconia or CPG®), titanium oxide, or synthetic polymers (e.g. polystyrene, polyvinylether, polyvinyl alcohol, monodisperse polyacrylate resin, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, etc.) and hydrogels of various compositions. In certain aspects, the solid support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of suitable polysaccharides include agar, agarose, dextran, starch, cellulose, pullulan, etc., and stabilized variants thereof. In some aspects, the solid support is made from agarose or a stabilized derivative thereof, such as Praesto®Pure, Praesto® Jetted A50, or polyvinyl divinyl benzene, silica, or control pore glass.

[0047] In other aspects the solid support may be a microchip, e.g., a silicon, silicon-glass, or gold microchip. In aspects, the solid support may be formed from nitrocellulose, paper, plastic, nylon, metal, or any combination of the foregoing. In some aspects, the solid support may be a plate or dish, such as a microtiter plate or dish.

[0048] Also provided are methods for removing contaminating dsRNA from a mixture comprising a target ssRNA product. In aspects, the methods comprise buffering a mixture comprising dsRNA and ssRNA to a pH of from 7.2 to 10 and adjusting a salt concentration to from about 0.3 to 2.5 M sodium chloride (NaCl) or potassium chloride (KC1), or from about 0.3 to 0.5 M NaCl or KC1, or similar high salt concentration, followed by passing the mixture through a chromatography column comprising an affinity matrix functionalized with the dsRNA-binding polypeptides described herein. Other salts may be used including ammonium sulfate, ammonium chloride, sodium sulfate, potassium sulfate, magnesium chloride, magnesium sulfate, etc. In aspects, the mixture is passed through the column at a flow rate sufficient to maintain a contact time of from about 15-30 seconds or more, e.g., about 15 to 40 seconds, about 15 to 60 seconds, about 15 to 90 seconds, about 30 to 90 seconds, about 30-180 seconds, or about 30 to 240 seconds. The methods may also include analysis of the column eluate for residual dsRNA, for example by a method comprising one or more of northern hybridization, including dot-blots, immuno-dot blots, and immuno-northern blots, as well as enzyme-linked immunosorbent assays (ELISA). In aspects, the methods described here provide a 1-3 log reduction in the amount of dsRNA compared to the amount present in the original mixture along with at least about 90% or greater recovery of the ssRNA with no negative impact on integrity of the ssRNA, as determined for example, by gel electrophoresis and/or capillary electrophoresis.

[0049] In aspects, an affinity chromatography matrix functionalized with the dsRNA-binding polypeptides or chimeric proteins described here may be subjected to a typical cleaning in place or “CIP” process without significant decrease in dsRNA-binding capacity. Typical CIP processes include subjecting the affinity matrix to conditions of alkaline pH, including for example 0.01-0.05 M NaOH, acidic pH, or high concentration of a chaotropic agent such as 2-8 M urea or 2-6 M guanidine hydrochloride. In some aspects, an affinity chromatography matrix functionalized with the dsRNA-binding polypeptides or chimeric proteins described here may retain at least 80%, preferably at least 90%, of its dsRNA-binding capacity following exposure to 0.01-0.05 M NaOH or 6 M guanidine hydrochloride for at least 10 hours, or at least 20 hours, or at least 30 hours, where dsRNA-binding capacity is measured as a percentage of the dynamic binding capacity (DBC) of the functionalized resin. In aspects, an affinity chromatography matrix functionalized with the dsRNA-binding polypeptides or chimeric proteins described here are provided as single-use columns.

[0050] Also provided are systems for purification of RNA in an RNA manufacturing process. In aspects, the RNA manufacturing system comprises an in vitro transcription (IVT) unit and an RNA purification unit which comprises at least one step of affinity chromatography utilizing an affinity chromatography matrix functionalized with the dsRNA-binding polypeptides or chimeric proteins described here to deplete contaminating dsRNA from a mixture of comprising a target ssRNA product in accordance with the methods described here.

[0051] The dsRNA-binding polypeptides and chimeric proteins described herein may be produced synthetically e.g., by liquid or solid phase chemical synthetic methods, and/or other techniques such as polymerase chain reaction (PCR) based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL). The dsRNA-binding polypeptides may also be produced recombinantly via expression in a host cell from polynucleotides encoding the polypeptides. Such polynucleotides may also be produced synthetically or using recombinant techniques based on the amino acid sequences of the dsRNA-binding polypeptides and chimeric proteins disclosed herein. It is understood that different nucleic acid sequences may be utilized to encode the same dsRNA-binding polypeptide or chimeric protein, for example where codon optimization is utilized to facilitate expression in a particular cell system. Methodologies and tools for constructing an optimized RNA sequence for protein expression are known in the art. See e.g., Strategies of codon optimization for high-level heterologous protein expression in microbial expression systems, Gene Reports 9:46-53 (2017); and “A new and updated resource for codon usage tables” Athey, J., Alexaki, A., Osipova, E. et al. A new and updated resource for codon usage tables. BMC Bioinformatics 18, 391 (2017).

[0052] Polynucleotides encoding the polypeptides described herein may comprise control elements such as promoters, enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, or may be inserted into an appropriate expression vector containing one or more such control elements for expression of the polynucleotides in a host cell such as a bacterial cell, yeast cell, insect cell, plant cell or mammalian cell. Examples of prokaryotic host expression systems, viral expression systems, yeast expression systems, plant expression systems, and mammalian expression systems are known in the art. Insertion of of the polynucleotides into a suitable expression vector can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, or in vivo recombination/genetic recombination techniques. Suitable expression vectors are commercially available and may include, for example, plasmid vectors, single and double-stranded phage vectors, or single and double-stranded RNA or DNA viral vectors. Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated viral particles using known techniques for infection and transduction. Alternatively, cell-free translation systems may also be used to produce the polypeptides.

[0053] In an exemplary embodiment, dsRNA-binding polypeptides are produced recombinantly in an appropriate expression system such as an E. Coli, Pichia Pastoris or in a mammalian cell system, using standard techniques. The recombinantly produced polypeptides are purified using multi-column chromatography. For example, histidine-tagged polypeptides may be purified using immobilized metal ion affinity chromatography (“IMAC”). The purity and identity of recombinant polypeptides may be assessed by a combination of gel electrophoresis, e.g., SDS-PAGE, reverse-phase ultra-high performance chromatography (“RP- UPLC”), quadrupole time-of-flight mass spectrometry, and size-exclusion chromatography (“SEC”).

[0054] It should be noted that the N-terminal methionine of recombinantly produced polypeptides may be cleaved during expression resulting in polypeptides lacking the N-terminal methionine, or a mixture of polypeptides which contain or lack the N-terminal methionine. The presence or absence of the N-terminal methionine does not impact the function of the polypeptides or chimeric proteins described herein. Accordingly, the N-terminal methionine should be considered optional in the amino acid sequences of the dsRNA-binding polypeptides and chimeric proteins described herein.

[0055] As discussed above, the affinity chromatography matrices provided herein comprise a solid support functionalized with a dsRNA-binding polypeptide or chimeric protein, as described herein. In aspects, the solid support is a discrete particle or bead. In some aspects, the bead is chemically activated for covalent attachment of the polypeptides, for example by disuccinimidyl carbonate, which may in turn by amidated, for example with excess ethylenediamine. In a subsequent step, bromoacetate may be conjugated to the aminated bead using a chemical coupling reagent such as l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) to which the polypeptide may be conjugated at room temperature, or at a temperature of from about 37 to 40 °C. In aspects, an affinity resin produced in this manner comprises dsRNA-binding polypeptide at a density of from about 5 to 30 g/L, as measured for example using a subtractive RP-HPLC method according to the following formula:

Actual Ligand Density = (Measured [ligand] in feed - Measured [ligand] in effluent).

[0056] The following examples describe work leading to a model dsRNA specific affinity resin that efficiently removes dsRNA from mixtures of dsRNA and ssRNA. The model affinity resins utilized in the following examples comprise recombinant dsRNA-binding polypeptides as described herein covalently coupled to an inert, insoluble polysaccharide bead which is an agarose or agarose derivative.

[0057] FIG. 1 shows a filter-plate screen of various conditions of salt concentration and pH in an experiment to identify optimal conditions for selective binding of dsRNA over ssRNA by an affinity chromatography matrix functionalized with a representative dsRNA-binding polypeptide, having a binding domain corresponding to SEQ ID NO: 6.

[0058] The pH range used was 6.5 to 10, from left to right: pH 6.5, 7.2, 8, 9, 9.5, and 10. Salt conditions were 0 to 2.5 M NaCl, top to bottom: 0, 0.5, 0.75, 1.25, 1.5, 2, and 2.5 M NaCl. Samples in the first six lanes included a mixture of ssRNA and dsRNA with the affinity resin. Samples in lanes 7-12 did not include the affinity resin.

[0059] The RNA mixture was incubated with the affinity matrix for 30 minutes with shaking followed by centrifugation. The amount of unbound dsRNA in the flow-through liquid was measured to assess performance of the resin in removing dsRNA from the sample. The dsRNA content was analyzed by immuno-dot blot using dsRNA specific monoclonal antibody. The amount of dsRNA remaining in each sample is indicated by dot intensity. As shown in the figure, the affinity resin effectively reduced the amount of dsRNA in the samples under conditions of pH 7.2-10 and 0.3-2.5 M NaCl. Similar results were obtained for an affinity chromatography matrix functionalized with a dsRNA-binding polypeptide having a binding domain corresponding to SEQ ID NO: 6.

[0060] FIG. 2 shows immuno-northern blot analysis of the pH 7.2 samples from the experiment in FIG. 1. Samples were subjected to gel electrophoresis and nucleic acid in each lane was visualized using a SYBR Safe nucleic acid stain (bottom panel in figure). After imaging, the gel was transferred to a positively charged nylon membrane and immunoblotted with the dsRNA specific J2 mAb (top panel in figure). A smear of high-molecular weight dsRNA is evident in all samples lacking affinity resin while the dsRNA is absent from all samples containing the resin except where salt concentration is zero. Removal of dsRNA increased with increasing salt concentrations.

[0061] FIG. 3 illustrates analysis of the dsRNA binding capacity of a test column. A 2.5 x 0.3 cm glass column was packed with an affinity resin comprising dsRNA-binding polypeptide as described herein and challenged with a 500 base-pair dsRNA (0.025 mg/ml concentration). Breakthrough of dsRNA was detected after 4.4 milliliters of sample application, which indicates a 10% breakthrough capacity of approximately 0.5 grams of dsRNA per liter of resin. Because the levels of dsRNA contaminants are typically 0.1-1% of the total ssRNA, a capacity of 0.5 grams per liter means that a 1 L column can process 500 grams of ssRNA with 0.1% dsRNA content, or 50 grams of ssRNA with a 1% dsRNA content. Thus, the dsRNA capacity demonstrated here is sufficient for bioprocessing operations. The top panel of the figure shows the chromatography trace of UV (255 nm) versus volume and an immunonorthern blot of the corresponding fraction on the bottom.

[0062] Affinity resins functionalized with each of 24 dsRNA-binding polypeptides were tested for their ability to discriminate ssRNA from dsRNA in a filter plate screen. Resin beads (5 ul) functionalized with each polypeptide were incubated with 100 ul of a mixture of ssRNA (1.8 kb firefly luciferase) and dsRNA (500 bp dsRNA). Each RNA was diluted to 27 ng/ul in a buffer consisting of 10 mM Tris pH 8, 100 mM NaCl and ImM EDTA. After 60 minutes incubation with shaking, the filter plate was centrifuged to collect the flow-through samples. The flow- through samples were analyzed on an agarose gel and band intensities (arbitrary units) were quantified with densitometry. The ratio of bound ssRNA to bound dsRNA bands is shown in FIG. 4 for resins functionalized with dsRNA-binding polypeptides.

[0063] A resin binding dsRNA with high specificity compared to ssRNA is indicated by a large ratio of ssRNA/dsRNA in this assay due to the relatively high ratio of ssRNA in the flowthrough, compared to dsRNA, for such a resin. Thus, the amount of ssRNA in the flow-through is high where the resin fails to bind ssRNA or binds it only weakly and the resulting ssRNA band intensity is also high. Where the resin also binds dsRNA with high affinity, the dsRNA in the flow-through will be low due to dsRNA having been bound by the resin, and the dsRNA band intensity will be correspondingly low. Thus, in FIG. 4, taller bars indicate resins with high selectivity for dsRNA over ssRNA. The data show that the resins functionalized with affinity ligands in accordance with the present invention effectively discriminated between dsRNA and ssRNA. Negative controls were “blank” resin beads, “b” in the figure, referring to resin beads not functionalized with ligand, and input sample, “in” in the figure, referring to the mixture of RNA before incubation with the affinity resin (data not shown). Bars 1-10 in FIG. 4 represent resins prepared with the following affinity ligands of Formula I, respectively: SEQ ID NO: 18, in which Di comprises a trimer of SEQ ID NO: 6, SEQ ID NO: 12, in which Di comprises SEQ ID NO: 5, SEQ ID NO: 14, in which Di comprises SEQ ID NO: 5, SEQ ID NO: 9, in which Di comprises SEQ ID NO: 2, SEQ ID NO: 8, in which Di comprises SEQ ID NO: 1, SEQ ID NO: 15, in which Di comprises SEQ ID NO: 6, SEQ ID NO: 10, in which Di comprises SEQ ID NO: 3, SEQ ID NO: 19, in which Di comprises SEQ ID NO: 7, SEQ ID NO: 11, in which Di comprises SEQ ID NO: 4, and SEQ ID NO: 17, in which Di comprises a monomer of SEQ ID NO: 6.

[0064] Affinity resins functionalized with two different ligands in accordance with the present invention, SEQ ID NO: 17 and SEQ ID NO: 18, were compared against a resin functionalized with a prior art ligand and a reference cellulose-based method for removal of dsRNA, described in Baiersdbrfer et al., Mol Ther Nucleic Acids 2019 Feb 27;15:26-35. This reference method is based on selective binding of dsRNA to cellulose in an ethanol-containing buffer and has been shown to remove 90% of the dsRNA contaminants. The prior art ligand was a Thermotoga maritima RNAse III corresponding to sequence number 3 of U.S. Patent No. 11,384,352. The experiment was performed using resin at a ligand density of 6.3 g/L. Binding was assayed using a mixture of dsRNAs ranging in size from 1,800 base-pairs (bp) to 80 bp. As shown in FIG. 5, the resins prepared with ligands described herein (SEQ ID NO: 17 and SEQ ID NO: 18) bound dsRNA with similar efficacy as the reference method of Baiersdorfer. In contrast, the resin functionalized with the prior art Thermotoga maritima RNAse III based ligand performed very poorly (SeqX in the figure).

Additional Embodiments

[0065] In one aspect, provided is an affinity chromatography matrix that includes a solid support and a double stranded RNA (dsRNA)-binding polypeptide covalently attached to the solid support, where the dsRNA-binding polypeptide includes from one to six RNA binding domains, each defined by a core sequence of from 70 to 200 amino acids, where the core sequence of each of the RNA binding domains is the same or different.

[0066] The affinity matrix may also include where the core sequence of each RNA-binding domain is independently 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: 7, SEQ ID NO: 13, and SEQ ID NO: 16, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0067] The affinity matrix may also include where the core sequence of each RNA-domain is independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 13, or SEQ ID NO: 16, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0068] The affinity matrix may also include where the core sequence of each RNA-domain is independently selected from SEQ ID NO: 13 or SEQ ID NO: 16, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0069] The affinity matrix may also include where the core sequence of each RNA-domain is independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, or an amino acid sequence having 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0070] The affinity matrix may also include where the dsRNA-binding polypeptide covalently attached to the solid support is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, or a polypeptide having an amino acid sequence with 90%, 95%, 97%, 98%, or 99% amino acid sequence identity thereto which retains dsRNA-binding activity.

[0071] The affinity matrix may also include where the solid support comprises dsRNA binding polypeptide at a density of from about 10-20 mg/mL, or about 15-20 mg/ml.

[0072] The affinity matrix may also include where the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

[0073] The affinity matrix may also include where each RNA-binding domain includes at a C- terminal end of its core sequence one or more of a spacer molecule, a reactive thiol- or nitrogen-containing amino acid, or a peptide, polypeptide, or protein tag. The affinity matrix may also include where the C-terminal spacer molecule is selected from an amino acid, a polypeptide of from 1-50 amino acids, or a non-polypeptide molecule, the C-terminal reactive thiol- or nitrogen-containing amino acid is cysteine or lysine, and the C-terminal tag is a polyhistidine.

[0074] The affinity matrix may also include where the affinity matrix has a dynamic binding capacity of at least about 5 mg/ml for dsRNA.

[0075] The affinity matrix may also include where the discrete polymeric particles are particles of a polysaccharide or synthetic polymer, optionally where the polysaccharide is agarose or cellulose, or a stabilized variant or derivative thereof.

[0076] Also provided are methods for depleting dsRNA from a mixture of dsRNA and ssRNA. In an aspect, provided is a method for depleting dsRNA from an aqueous mixture includes dsRNA and ssRNA, the method comprising contacting the mixture with an affinity matrix as described herein. In aspects, the method may also include where the contacting is performed under conditions suitable for binding of the dsRNA to the affinity matrix and collecting the ssRNA in the flow-through from the affinity matrix.

[0077] The method may also include where the contacting occurs as the mixture flows through a chromatography column includes the affinity matrix. The method may also include where the method includes buffering the mixture includes dsRNA and ssRNA to a pH of from 7.2 to 10 and adjusting a salt concentration of the mixture to from about 0.3 to 2.5 M sodium chloride (NaCl) prior to contacting the mixture with the affinity matrix. The method may also include where the method includes an additional step of eluting the dsRNA from the affinity matrix following the step of collecting the ssRNA. The method may also include where the method includes a cleaning in place step following elution of the dsRNA from the affinity matrix, includes contacting the affinity matrix with an alkaline solution includes from 0.01- 0.05 M NaOH or 6 M guanidine hydrochloride for a period of at least 10 hours, optionally at least 20 hours or at least 30 hours.

[0078] Also provided are polynucleotides encoding the dsRNA polypeptides described herein. In one aspect, provided is a polynucleotide encoding the dsRNA-binding polypeptide of any one of SEQ ID NOs 1-19.

[0079] Also provided are recombinant cells comprising a polynucleotide encoding the dsRNA-binding polypeptide as described herein. In one aspect, provided is a recombinant cell or viral particle comprising a polynucleotide encoding the dsRNA-binding polypeptide of any one of SEQ ID NOs 1-19. [0080] Also provided are systems for RNA manufacture. In one aspect, provided is a system for RNA manufacture comprising an in vitro transcription unit, an RNA purification unit, and a ssRNA purification unit, where the ssRNA purification unit includes an affinity chromatography apparatus comprising an affinity matrix as described herein. In aspects, the RNA purification unit comprises an RNA affinity column, optionally where the RNA affinity column is an oligo(dT)-cellulose column or a poly(U)-Sepharose column. The system may also include where the ssRNA purification unit is placed before or after the RNA affinity column.

[0081] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims. [0082] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0083] Various features of a process system may be used independently of, or in combination, with each other. It will be appreciated that a system as disclosed herein may be embodied in different forms and should not be construed as limited to the illustrated embodiments of the figures.

[0084] It should be understood that, as described herein, an “embodiment” (such as illustrated in the accompanying Figures) may refer to an illustrative representation of an environment or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied. However such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. In addition, it will be appreciated that while the Figures may show one or more embodiments of concepts or features together in a single embodiment of an environment, article, or component incorporating such concepts or features, such concepts or features are to be understood (unless otherwise specified) as independent of and separate from one another and are shown together for the sake of convenience and without intent to limit to being present or used together. For instance, features illustrated or described as part of one embodiment can be used separately, or with one or more other features to yield a still further embodiment. Thus, it is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0085] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and/or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0086] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by +/- 10%.

[0087] In the claims, the term “comprises/comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0088] The term “polypeptide” refers to any linear molecular chain of two or more amino acids linked by peptide bonds and does not refer to a specific length of the product. Thus, “peptides”, “amino acid chain,” or any other term used to refer to a chain of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. [0089] The term “percent (%) identity”, in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Percent identity may be determined using a computer algorithms such as the Basic Local Alignment Search Tool (“BLAST”) or a related tool, including other BLAST-based tools available at the US National Library of Medicine, National Center for Biotechnology Information website. The BLAST and related algorithms have been described, for example, in Altschul et al., 1990, J. Mol. Biol. 215:3, 403-410; and Altschul et al., 1991, Nucleic Acids Res. 25: 17, 3389-402.

[0090] The term “substitution” in the context of an amino acid substitution refers to an exchange of an amino acid at a particular position in a polypeptide sequence with a different amino acid. Similarly, the term “deletion” in the context of an amino acid deletion refers to the removal of an amino acid at a particular position in a polypeptide sequence; and the term “insertion” in the context of amino acid insertion refers to the addition of an amino acid to the polypeptide sequence.

[0091] The term “chromatography” refers to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules (e.g., immunoglobulins) from other molecules (e.g. contaminants or other immunoglobulins) in a sample. A liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase which may be referred to herein interchangeably as a matrix or resin. 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. The term “affinity chromatography” refers to a specific type of chromatography in which a ligand having a specific affinity for a target molecule is coupled to the stationary phase. The ligand interacts with the target molecule in the mobile phase thereby separating it from the mobile phase. Generally, the target molecule is eluted from the stationary phase in a separate step. The terms “affinity matrix” or “matrix” or “resin” are used interchangeably herein to refer to the solid support functionalized with an affinity ligand as described herein, i.e., a dsRNA-binding polypeptide as described herein.

[0092] A “conservative” amino acid substitution is one in which one amino acid residue is replaced with another having similar side chain chemistry and/or size. Families of amino acid residues having similar side chain chemistry have been defined in the art. For example, those having basic side chains, e.g., lysine (K), arginine (R), histidine (H); acidic side chains, e.g., aspartic acid (D), glutamic acid (E); uncharged polar side chains, e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C); nonpolar side chains, e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); beta-branched side chains, e.g., threonine (T), valine (V), isoleucine (I); and aromatic side chains, e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H).

Informal Sequence Listing

Claims

CLAIMS What is claimed is:

1. An affinity ligand of Formula I, or a multimer thereof:

L1-D1-L2-DS-L3-DT-L4 (Formula I) wherein

Li, L2, L3, and L4 are each independently absent or a linker;

Di is a dsRNA-binding domain comprising an amino acid sequence 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing;

Ds is an optional structural domain; and

DT is an optional C-terminal tag domain.

2. An affinity chromatography matrix comprising a solid support and a double stranded RNA (dsRNA)-binding polypeptide ligand covalently attached to the solid support, wherein the ligand comprises from one to six dsRNA-binding domains, wherein each dsRNA-binding domain is defined by a core sequence of from 60-200 amino acids, optionally from 70-200 amino acids, and wherein the core sequence is the same or different in each of the from one to six dsRNA-binding domains.

3. The affinity chromatography matrix of claim 2, wherein each dsRNA-binding domain comprises at a C-terminal end of its core sequence one or more of a spacer molecule, a reactive thiol- or nitrogen-containing amino acid, a structural domain, or a peptide, polypeptide, or protein tag.

4. The affinity chromatography matrix of claim 3, wherein the C-terminal spacer molecule is selected from an amino acid, a polypeptide of from 1- 60 amino acids, or a non-polypeptide molecule; the C-terminal reactive thiol- or nitrogen-containing amino acid is cysteine or lysine; and the C-terminal tag is a cysteine.

5. The affinity chromatography matrix of any one of claims 2 to 4, wherein the core sequence comprises a 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing.

6. The affinity chromatography matrix of any one of claims 2 to 5, wherein the ligand is a polypeptide of Formula I, or a multimer thereof:

L1-D1-L2-DS-L3-DT-L4 (Formula I) wherein

Li, L2, L3, and L4 are each independently absent or a linker;

Di is a dsRNA-binding domain comprising a 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, SEQ ID NO: 6 and SEQ ID NO: 7, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity to any one of the foregoing;

Ds is an optional structural domain; and

Dr is an optional C-terminal tag domain.

7. The affinity chromatography matrix of any one of claims 2 to 6, or the affinity ligand of claim 1, wherein the ligand comprises from two to four dsRNA-binding domains, optionally wherein the ligand is a homomultimer or a heteromultimer with respect to the dsRNA-binding domains.

8. The affinity chromatography matrix of any one of claims 2 to 7, or the affinity ligand of claim 1, wherein the ligand consists of SEQ ID NO: 20, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

9. The affinity chromatography matrix of any one of claims 2 to 7, or the affinity ligand of claim 1, wherein the ligand consists of SEQ ID NO: 21, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

10. The affinity chromatography matrix of any one of claims 2 to 7, or the affinity ligand of claim 1, wherein the ligand consists of SEQ ID NO: 24, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

11. The affinity chromatography matrix of any one of claims 2 to 7, or the affinity ligand of claim 1, wherein the ligand consists of an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

12. The affinity chromatography matrix of any one of claims 2 to 7, or the affinity ligand of claim 1, wherein the ligand consists of an amino acid sequence of SEQ ID NO: 26, or a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 93%, 95%, 97%, or 98% amino acid sequence identity thereto which retains dsRNA-binding activity.

13. An affinity chromatography matrix comprising a solid support and the affinity ligand as defined in claim 1 or as defined in any one of claims 2 to 12, covalently attached to the solid support.

14. The affinity chromatography matrix of any one of claims 2 to 13, wherein the solid support comprises dsRNA binding polypeptide at a density of from about 10-20 mg/mL, or about 15-20 mg/ml.

15. The affinity chromatography matrix of claim 14, wherein the affinity matrix has a dynamic binding capacity of at least about 5 mg/ml for dsRNA.

16. The affinity chromatography matrix of any one of claims 2 to 15, wherein the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

17. The affinity chromatography matrix of claim 16, wherein the discrete polymeric particles are particles of a polysaccharide or synthetic polymer, optionally wherein the polysaccharide is agarose or cellulose, or a stabilized variant or derivative thereof, and the synthetic polymer is a polymethacrylate polymer.

18. A method for depleting dsRNA from an aqueous mixture comprising dsRNA and ssRNA, the method comprising contacting the mixture with the affinity chromatography matrix of any one of claims 2 to 17 under conditions suitable for binding of the dsRNA to the affinity matrix, and collecting the ssRNA in the flow-through from the affinity matrix.

19. The method of claim 18, wherein the contacting occurs as the mixture flows through a chromatography column comprising the affinity chromatography matrix.

20. The method of claim 18 or 19, wherein the method comprises buffering the mixture comprising dsRNA and ssRNA to a pH of from 7.2 to 10 and adjusting a salt concentration of the mixture to from about 0.3 to 2.5 M sodium chloride (NaCl) prior to contacting the mixture with the affinity chromatography matrix.

21. The method of any one of claims 18 to 20, wherein the method comprises an additional step of eluting the dsRNA from the affinity chromatography matrix following the step of collecting the ssRNA.

22. The method of any one of claims 18 to 21, wherein the method comprises a cleaning in place step following elution of the dsRNA from the affinity chromatography matrix, comprising contacting the affinity matrix with an alkaline solution comprising from 0.01-0.05 M NaOH or 6 M guanidine hydrochloride for a period of at least 10 hours, optionally at least 20 hours or at least 30 hours.

23. A polynucleotide encoding the dsRNA-binding polypeptide of any one of SEQ ID NOs 1- 26.

24. A recombinant cell or viral particle comprising a polynucleotide encoding the dsRNA- binding polypeptide of any one of SEQ ID NOs 1-26.

25. A system for RNA manufacture comprising an in vitro transcription unit, an RNA purification unit, and a ssRNA purification unit, wherein the ssRNA purification unit comprises an affinity chromatography apparatus comprising the affinity matrix of any one of claims 2 to 17.

26. The system of claim 25, wherein the RNA purification unit comprises an RNA affinity column, optionally wherein the RNA affinity column is an oligo(dT)-cellulose column or a poly(U)-Sepharose column.

27. The system of claim 26, wherein the ssRNA purification unit is placed before or after the RNA affinity column.