A method for purifying single-stranded RNA

Abstract

The present disclosure relates to improved therapeutic RNA compositions. More specifically, the present disclosure relates to improved methods for purifying therapeutic RNA and related therapeutic RNA preparations.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 210,101, filed June 14, 2021, which is incorporated by reference herein in its entirety.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is BLUE-136_PC_SL.txt. The text file is 39,804 bytes in size, was created on June 9, 2022, and is being submitted electronically via EFS-Web simultaneously with the filing of this application. [Background technology]

[0003] The present disclosure relates to improved RNA compositions. More specifically, the present disclosure relates to improved methods for purifying therapeutic RNA and related therapeutic RNA preparations.

[0004] Description of related fields Over the past 30-40 years, there has been a gradual emergence of RNA-based technologies, which have recently gained increasing attention as new methods for RNA delivery have been developed and proven effective in vivo. For example, RNAi (e.g., siRNA, shRNA, or miRNA), ribozymes, aptamers, and related technologies have been used to reduce the expression or modulate the activity of disease-related proteins. In other cases, RNA has been used to express proteins either in vitro, ex vivo, or in vivo for therapeutic purposes. However, double-stranded RNA (dsRNA) can be toxic to cells in certain circumstances. There remains a need to develop improved methods for the specific removal of dsRNA from RNA preparations, especially for in vivo therapeutic applications. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure generally relates, in part, to an RNA purification method / process that includes a step of removing dsRNA. In some embodiments, the method / process includes one or more steps of oligo-dT purification and a step of dsRNA removal.

[0006] In one aspect, an RNA purification process is provided that includes contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen-binding fragment thereof that binds to dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and purifying the single-stranded RNA.

[0007] In another aspect, an RNA purification process is provided that includes contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen-binding fragment thereof that binds to dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and purifying the single-stranded RNA, thereby producing a therapeutic RNA.

[0008] In another aspect, an RNA purification process is provided that includes contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) encoding a nuclease with an antibody or antigen-binding fragment thereof that binds the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and purifying the single-stranded RNA, wherein the rate of editing nuclease is increased compared to the rate of editing of a nuclease encoded by RNA that is not contacted with the antibody that binds the dsRNA.

[0009] In another aspect, a process for RNA purification is provided, comprising contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen-binding fragment thereof that binds dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and purifying the single-stranded RNA, wherein the RNA is less immunogenic and / or toxic when administered to a cell or subject than the immunogenic and / or toxic of the RNA administered to the cell or subject when the RNA is not contacted with the antibody that binds dsRNA. In some embodiments, the subject is a human.

[0010] In various embodiments, the single stranded RNA is a single stranded circular RNA, a single stranded mRNA, or a single stranded non-coding RNA.

[0011] In various embodiments, the single stranded RNA is polyadenylated and / or the process includes a step of polyadenylation prior to contacting the sample with an antibody, or antigen-binding fragment thereof, that binds the dsRNA.

[0012] In various embodiments, the process includes contacting a polyadenylated RNA sample with a first oligonucleotide dT (oligo dT) probe that binds to the polyadenylated RNA, and removing unbound RNA from the sample before contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA.

[0013] In various embodiments, the process further comprises contacting the polyadenylated RNA with a second oligonucleotide dT probe after contacting with an antibody or antigen-binding fragment thereof that binds the dsRNA.

[0014] In various embodiments, the RNA sample is obtained de novo through chemical synthesis, hi some embodiments, the RNA sample is obtained from an in vitro transcription reaction.

[0015] In various embodiments, the cytotoxicity, as measured by impedance, of the purified RNA when administered to a cell is less than the cytotoxicity of the RNA administered to a cell when the RNA is not contacted with the dsRNA and / or the second oligo-dT-binding antibody.

[0016] In various embodiments, the first and / or second oligonucleotide dT probe is attached to a surface. In some embodiments, the first and / or second oligonucleotide dT probe is covalently attached to a surface.

[0017] In various embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample. In some embodiments, the RNA is obtained from an in vitro transcription reaction and is co-transcriptionally capped. In some embodiments, the cap is Cap0 or Cap1. In some embodiments, the cap is an ARCA cap or a modified ARCA cap. In some embodiments, the RNA in the sample is capped at its 5' end using a capping enzyme, guanosine triphosphate, and S-adenosyl-L-methionine. In certain embodiments, the capping enzyme is vaccinia guanylyltransferase. In some embodiments, the capping comprises guanosine triphosphate. In some embodiments, the capping comprises S-adenosyl-L-methionine. In some embodiments, the capping comprises 2'-O-methyltransferase.

[0018] In various embodiments, the antibody or antigen-binding fragment thereof that binds dsRNA is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv"), and single domain antibody (sdAb, camelid VHH, nanobody). In some embodiments, the antibody or antigen-binding fragment thereof that binds dsRNA is a monoclonal antibody. In some embodiments, the antibody is selected from the group consisting of J2, J5, K1, K2, 1D3, CABT-B212, and 9D5. In a particular embodiment, the antibody is J2.

[0019] In various embodiments, the sample is contacted with at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%, at least about 5.5 mol%, at least about 6 mol%, at least about 6.5 mol%, at least about 7 mol%, at least about 7.5 mol%, at least about 15 mol%, at least about 30 mol%, or at least about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with at least about 1.5 mol%, at least about 7.5 mol%, at least about 15 mol%, at least about 30 mol%, or at least about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with at least about 7.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 15 mol%, about 30 mol%, or about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 7.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 2 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 2.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 3 mol% to about 60 mol% of the antibody relative to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 3.5 mol% to about 60 mol% of the antibody relative to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 4 mol% to about 60 mol% of the antibody relative to the total moles of RNA in the sample.In some embodiments, the sample is contacted with about 4.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 5.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 6 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 6.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 7 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 7.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 15 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 30 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 30 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 15 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 7.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 7 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 6.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 6 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 5.5 mol% of the antibody relative to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 5 mol% of the antibody relative to the total moles of RNA in the sample.In some embodiments, the sample is contacted with about 1.5 mol% to about 4.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 4 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 3.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 3 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 2.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the sample is contacted with about 1.5 mol% to about 2 mol% of the antibody compared to the total moles of RNA in the sample.

[0020] In various embodiments, the dsRNA:antibody complex is separated from the single-stranded RNA by antibody-based affinity chromatography. In some embodiments, the antibody-based affinity chromatography comprises a 1 ml column. In some embodiments, the antibody-based affinity chromatography comprises a 5 ml column. In some embodiments, the antibody-based affinity chromatography comprises a 10 ml column.

[0021] In various embodiments, the process includes a step of plasmid digestion prior to the step of IVT.

[0022] In various embodiments, the process further comprises treating the sample with DNase to remove residual plasmid DNA template, hi some embodiments, the DNase treatment step occurs after the IVT step and / or after the capping step.

[0023] In various embodiments, the process further comprises one or more steps of ultrafiltration / diafiltration (UF / DF), in some embodiments, the UF / DF step is after a plasmid digestion step, an in vitro transcription step, a capping reaction step, or an affinity chromatography step (e.g., dT or J2).

[0024] In various embodiments, the process further comprises a step of terminal sterile filtration, hi some embodiments, the step of terminal sterile filtration comprises filtration through a 0.22 μm filter.

[0025] In various embodiments, the nuclease is an endonuclease or an exonuclease. In some embodiments, the nuclease is a homing endonuclease, a megaTAL, a CRISPR-associated nuclease, a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN). In some embodiments, the CRISPR-associated nuclease is Cas9 or a variant thereof.

[0026] In various embodiments, the level of aspartate aminotransferase enzyme (AST) in a subject administered the purified RNA is lower than the level of AST in a subject administered purified RNA that has not been contacted with dsRNA and / or an antibody that binds to the second oligo-dT.

[0027] In various embodiments, the IL-6 level in a subject administered the purified RNA is lower than the IL-6 level in a subject administered purified RNA that has not been contacted with a dsRNA and / or an antibody that binds to the second oligo-dT.

[0028] In various embodiments, the level of MCP-1 in a subject administered the purified RNA is lower than the level of MCP-1 in a subject administered purified RNA that has not been contacted with dsRNA and / or an antibody that binds to the second oligo-dT. [Brief description of the drawings]

[0029] [Figure 1A] 1 illustrates different unit operations for an exemplary RNA purification method. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 2A] FIG. 1 shows dsRNA dot blot analysis of purified mRNA using different chromatography column volumes to remove excess anti-dsRNA antibodies. [Figure 2B] % dsRNA content in samples of purified mRNA using different chromatography column volumes to remove excess anti-dsRNA antibodies is shown. [Figure 2C] 1 shows a dot blot analysis of purified mRNA using a secondary fluorescent label to directly blot residual anti-dsRNA antibodies. [Figure 3A] The % dsRNA content in samples of purified mRNA using different amounts of anti-dsRNA antibody is shown. [Figure 3B] Same as above. [Figure 3C] FIG. 1 shows the in vitro cytotoxicity of purified mRNA using different amounts of anti-dsRNA antibody. [Figure 3D] Same as above. [Figure 4A] The % full length mRNA in samples purified by different methods is shown. [Figure 4B] The % dsRNA content in samples purified by different methods is shown. [Figure 4C] FIG. 1 shows the in vitro cytotoxicity of mRNA samples purified by different methods. [Figure 4D] Shown is the % INDEL fold change after in vivo editing with PCSK9 megaTAL and Trex2 encoded by mRNA purified by different methods. [Figure 4E] 1 shows the in vivo toxicity of PCSK9 megaTAL and Trex2 mRNA purified by different methods. [Figure 4F] 1 shows the degree of immunogenicity (cytokine / chemokine release) induced by PCSK9 megaTAL and Trex2 mRNA purified by different methods. [Figure 5A] The % full length mRNA in samples purified by different methods is shown. [Figure 5B] The % dsRNA content in samples purified by different methods is shown. [Figure 5C] FIG. 1 shows the in vitro cytotoxicity of mRNA samples purified by different methods. [Figure 5D] INDEL% after ex vivo editing with PD-1 megaTAL encoded by mRNA purified by different methods is shown. [Figure 5E] 6 shows the % PD-1 surface expression after ex vivo editing with PD-1 megaTALs encoded by mRNA purified by different methods. [Figure 6A] The dsRNA content (%) between different RNA purification methods is shown. [Figure 6B] 1 shows the correlation between cellular cytotoxicity and % dsRNA content of RNA preparations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Brief description of sequence identifiers SEQ ID NO:1 is the TCRα megaTAL DNA sequence. SEQ ID NO:2 is the TCRα megaTAL RNA sequence. SEQ ID NO:3 is the TCRα megaTAL RNA sequence. SEQ ID NO:4 is the PD1 megaTAL DNA sequence. SEQ ID NO:5 is the PD1 megaTAL RNA sequence. SEQ ID NO:6 is the PD1 megaTAL RNA sequence. SEQ ID NO:7 is the PCSK9 megaTAL DNA sequence. SEQ ID NO:8 is the PCSK9 megaTAL RNA sequence. SEQ ID NO:9 is the PCSK9 megaTAL RNA sequence. SEQ ID NO: 10 is the Trex2 DNA sequence. SEQ ID NO: 11 is the Trex2 RNA sequence. SEQ ID NO: 12 is the Trex2 RNA sequence.

[0031] In the above sequences, X refers to any amino acid, if present, or to the absence of an amino acid. [Mode for carrying out the invention]

[0032] A. Overview The present disclosure relates generally, in part, to improved methods of RNA purification and preparation of RNA compositions. More specifically, the present disclosure relates to improved methods for separating double-stranded RNA (dsRNA) from therapeutic single-stranded RNA and related therapeutic single-stranded RNA compositions. The RNA preparations can be for in vitro, ex vivo, or in vivo use. Without wishing to be bound by any particular theory, the inventors have discovered that RNA purification using anti-dsRNA antibodies (e.g., antibody-based affinity chromatography) is surprisingly effective in purifying single-stranded RNA and reducing the immunogenicity and cytotoxicity of RNA delivered to cells ex vivo and in vivo. In certain embodiments, the RNA purification method includes one or more steps of oligo-dT purification and anti-dsRNA antibody-based removal.

[0033] Thus, the problems of RNA immunogenicity and toxicity are resolved by utilizing anti-dsRNA antibody removal and / or oligo-dT purification, as further described herein. RNA purified using the compositions and methods contemplated in certain embodiments is suitable for use in in vitro, ex vivo, or in vivo applications.

[0034] In one aspect, an RNA purification process is provided that includes contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen-binding fragment thereof that binds dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and purifying the RNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process includes capping the RNA in the sample. In some embodiments, the process includes contacting the RNA sample with one or more oligonucleotide dT (oligo dT) probes that bind polyadenylated RNA and removing unbound RNA from the sample.

[0035] In another aspect, a process for RNA purification is provided, comprising contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA, removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA, and purifying the single-stranded polyadenylated RNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process comprises a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample.

[0036] In another aspect, a process for RNA purification is provided, comprising contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds the dsRNA to form a dsRNA:antibody complex, and removing the dsRNA:antibody complex from the sample, thereby purifying the single-stranded polyadenylated RNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process includes capping the RNA in the sample.

[0037] In another aspect, a process for RNA purification is provided, comprising contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and contacting the sample with a second oligonucleotide dT probe to capture the single-stranded polyadenylated RNA, thereby purifying the single-stranded polyadenylated RNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process includes capping the RNA in the sample.

[0038] In certain embodiments, the RNA is a therapeutic RNA (e.g., an mRNA). In certain embodiments, the RNA encodes a therapeutic polypeptide.

[0039] In another aspect, a process for increasing the efficiency of editing nucleases is provided, comprising contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA, removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA, and purifying the single-stranded polyadenylated RNA, wherein the rate of editing nucleases is increased compared to the rate of editing of nucleases encoded by RNA that is not contacted with the antibody that binds to the dsRNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process includes capping the RNA in the sample.

[0040] In another aspect, a process for increasing the efficiency of editing nucleases is provided, comprising contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA, removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, and removing the dsRNA:antibody complex from the sample, wherein the rate of editing nucleases is increased compared to the rate of editing of nucleases encoded by RNA that is not contacted with the antibody that binds to the dsRNA. In various embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process includes capping the RNA in the sample.

[0041] In another aspect, a process is provided for increasing the efficiency of editing nucleases, comprising contacting an RNA sample comprising single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and contacting the sample with a second oligonucleotide dT probe to capture the single-stranded polyadenylated RNA, wherein the rate of editing nucleases is increased compared to the rate of editing of nucleases encoded by RNA not contacted with the dsRNA and / or the antibody that binds to the second oligo dT. In various embodiments, the single-stranded RNA is a single-stranded circular RNA, a single-stranded mRNA, or a single-stranded non-coding RNA. In some embodiments, the single stranded RNA is polyadenylated and / or the process comprises a step of polyadenylation, hi some embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample.

[0042] In various embodiments, the nuclease is an endonuclease or an exonuclease. In some embodiments, the endonuclease is a homing endonuclease, a megaTAL, a CRISPR-associated nuclease (e.g., Cas9 and its variants), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN).

[0043] In another aspect, a process is provided for reducing the immunogenicity and / or toxicity of RNA administered to a cell or subject, comprising contacting an RNA sample comprising single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds the polyadenylated RNA, removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds the dsRNA, and purifying the single-stranded polyadenylated RNA, wherein the immunogenicity and / or toxicity of the RNA when administered to the cell or subject is less than the immunogenicity and / or toxicity of the RNA when administered to the cell or subject when the RNA is not contacted with the antibody that binds the dsRNA. In various embodiments, the single-stranded RNA is a single-stranded circular RNA, a single-stranded mRNA, or a single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample.

[0044] In another aspect, a process is provided for reducing the immunogenicity and / or toxicity of RNA administered to a cell or subject, comprising contacting an RNA sample comprising single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, and removing the dsRNA:antibody complex from the sample, wherein the immunogenicity and / or toxicity of the RNA when administered to the cell or subject is less than the immunogenicity and / or toxicity of mRNA administered to the cell or subject when the RNA is not contacted with the antibody that binds to the dsRNA. In various embodiments, the single-stranded RNA is a single-stranded circular RNA, a single-stranded mRNA, or a single-stranded non-coding RNA. In some embodiments, the single-stranded RNA is polyadenylated and / or the process includes a step of polyadenylation. In some embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample.

[0045] In another aspect, a process is provided for reducing the immunogenicity and / or toxicity of RNA administered to a cell or subject, comprising contacting an RNA sample comprising single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and contacting the sample with a second oligonucleotide dT probe to capture the single-stranded polyadenylated RNA, wherein the immunogenicity and / or toxicity of the RNA when administered to the cell or subject is less than the immunogenicity and / or toxicity of the RNA when administered to the cell or subject when the RNA is not contacted with the antibody that binds the dsRNA. In various embodiments, the single-stranded RNA is a single-stranded circular RNA, a single-stranded mRNA, or a single-stranded non-coding RNA. In some embodiments, the single stranded RNA is polyadenylated and / or the process comprises a step of polyadenylation, hi some embodiments, the RNA in the sample is capped and / or the process comprises capping the RNA in the sample.

[0046] In any of the embodiments contemplated herein, the anti-dsRNA antibody is selected from the group consisting of J2, J5, K1, K2, 1D3, CABT-B212, and 9D5, or a functional derivative or fragment thereof. In certain embodiments, the anti-dsRNA antibody is J2.

[0047] In any of the embodiments, the methods, procedures, or processes contemplated herein may include additional steps, such as plasmid linearization / digestion, in vitro transcription, diafiltration, ultrafiltration, and final filtration.

[0048] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, which are cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985), Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY)、Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK、Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)、Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991)、Oligonucleotide Synthesis(N.Gait,Ed.,1984)、Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985)、Transcription and Translation(B.Hames & S.Higgins,Eds.,1984)、Animal Cell Culture(R.Freshney,Ed.,1986)、Perbal,A Practical Guide to Molecular Cloning(1984)、Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH)、PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press)、Immobilized Cells And Enzymes(IRL Press,1986)、the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.)、Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.Please refer to research papers in specialist journals such as Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0049] B. Definition Before describing the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test specific embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0051] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more than one element.

[0052] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0053] The term "and / or" should be understood to mean either one or both of the alternatives.

[0054] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0055] In one embodiment, ranges such as, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each value subsumed within the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0056] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces an effect, e.g., a physiological effect, that is approximately the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0057] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to everything preceding the word "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to any elements listed after the phrase, and other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that there are no other elements that materially affect the activity or action of the recited elements.

[0058] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. As such, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Moreover, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the forward recitation of a feature in an embodiment serves as a basis for excluding the feature in a particular embodiment.

[0059] The term "ex vivo" generally refers to activities that occur at a location outside of an organism, such as experiments or measurements performed in an artificial environment outside of the organism, preferably with minimal changes to natural conditions, or experiments or measurements on living tissue. In certain embodiments, "ex vivo" procedures involve living cells or tissues taken from an organism and cultured or conditioned in a laboratory setup, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours depending on the circumstances. In certain embodiments, such tissues or cells can be collected, frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures that last for several days or more using living cells or tissues are typically considered to be "in vitro", although in certain embodiments, the term can be used interchangeably with ex vivo.

[0060] The term "in vivo" generally refers to activities that occur inside an organism. In one embodiment, the genome of a cell is manipulated, edited, or modified in vivo.

[0061] An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by vehicle or control.

[0062] The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a reduction that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) than the response produced by vehicle or control.

[0063] "Sustained" or "maintained" or "no change" or "no substantial change" or "no substantial decrease" refers to a response that is not significantly or measurably different from a baseline response, vehicle, or control.

[0064] The terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" as used herein describe the binding of one molecule to another molecule with a binding affinity higher than background binding, such as, for example, an antibody binding to double-stranded RNA (dsRNA), a nucleotide (e.g., oligo-dT) binding to a poly(A) tail, or a meganuclease (or binding domain) binding to a target site. An antibody, nucleotide, or binding domain may have a binding affinity of, for example, about 10 5 M -1 Affinity or K a (i.e., the equilibrium association constant for the particular binding interaction has units of 1 / M). In certain embodiments, a binding domain "specifically binds" to another molecule (e.g., DNA, RNA, or a polypeptide) if it binds or associates with the molecule with a constant .gamma. 6 M -1 , 10 7 M -1, 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than K a A "high affinity" binding domain has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or more than K a refers to those binding domains having the

[0065] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe the preferential binding of one molecule to a target molecule in the presence of multiple off-target molecules (on-target binding). In certain embodiments, the anti-dsRNA antibody or fragment thereof selectively binds to dsRNA about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the anti-dsRNA antibody binds to single-stranded RNA (ssRNA).

[0066] The term "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region or a fragment thereof that specifically recognizes and binds to an epitope of one or more antigens, such as peptides, lipids, polysaccharides, or nucleic acids containing antigenic determinants, such as those recognized by immune cells. In some embodiments, the antibody is an anti-dsRNA antibody. In certain embodiments, the anti-dsRNA antibody and the dsRNA form a dsRNA:antibody complex.

[0067] The term "antibody" encompasses any naturally occurring, recombinant, modified or engineered immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. The term thus refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. An antibody may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. An antibody or antigen-binding portion thereof may be produced in a hybridoma, by recombinant DNA techniques, or by enzymatic or chemical cleavage of an intact antibody.

[0068] The term "antigen-binding fragment" or "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., dsRNA). Antigen-binding fragments include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen forming a complex. In some embodiments, an antigen-binding portion of an antibody can be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the antibody variable and optionally constant domains.

[0069] An "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment.

[0070] As used herein, a "gene of interest" or a "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or protein of interest. Depending on the context, a gene of interest may refer to a gene of interest in a deoxyribonucleic acid, e.g., a DNA template that can be transcribed into an RNA transcript, or a gene of interest in a ribonucleic acid, e.g., an RNA transcript that can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide of interest. As described in more detail below, polypeptides of interest include, but are not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, endonucleases, exonucleases, and the like.

[0071] As used herein, the term "operably linked" refers to a juxtaposition in which the described components are in a relationship such that they can function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide encoding a gene of interest, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence. For example, a gene of interest operably linked to an RNA polymerase promoter allows for the transcription of the gene of interest.

[0072] As used herein, the terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably according to their conventional meaning, i.e., as amino acid sequences, unless otherwise specified. Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides in one or more amino acid substitutions, deletions, additions, and / or insertions. Such variants may be of natural origin or may be synthetically produced, e.g., by modifying one or more amino acids of the polypeptide sequence.

[0073] As used herein, "poly A tail," "poly(A) tail," or "poly(A)" refers to a chain of adenine nucleotides. The term may refer to a poly(A) tail that is added to an RNA transcript, or may refer to a poly(A) tail that is already present at the 3' end of an RNA transcript (e.g., a DNA-encoded poly(A) tail). As described in more detail below, poly(A) tails are typically between 5 and 300 nucleotides in length (SEQ ID NO: 13).

[0074] The term "polynucleotide" is interchangeable with the term "nucleic acid" and includes any compound and / or substance comprising a polymer of nucleotides. Thus, the term "polynucleotide" or "nucleic acid" includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA having a β-D-ribostructure, α-LNA having an α-L-ribostructure (diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), or hybrids thereof.

[0075] Additional definitions are set forth throughout this disclosure.

[0076] C. Method As discussed throughout this disclosure, the inventors have recognized that dsRNA is responsible for increased immunogenicity and increased cytotoxicity in RNA compositions, particularly in the context of in vivo therapeutic RNA applications. Furthermore, the inventors have surprisingly discovered improved methods / processes for removing dsRNA from RNA preparations.

[0077] Whether the method is for RNA purification generally or for a specific application (e.g., a process for improving in vivo therapeutic mRNA treatment or in vivo gene editing), the method includes contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen-binding fragment thereof that binds to dsRNA and purifying the single-stranded RNA.

[0078] In one aspect, the process includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA, and purifying the single-stranded polyadenylated RNA.

[0079] In another aspect, a process includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, and removing the dsRNA:antibody complex from the sample, thereby purifying the single-stranded polyadenylated RNA.

[0080] In yet another aspect, the process includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and contacting the sample with a second oligonucleotide dT probe to capture single-stranded polyadenylated mRNA, thereby purifying single-stranded capped polyadenylated RNA.

[0081] As further described below, the method may include other steps, such as plasmid linearization / digestion, in vitro transcription, polyadenylation, capping, diafiltration, ultrafiltration, and final filtration. In some embodiments, the RNA may be for use in in vitro, ex vivo, or in vivo methods. In some embodiments, the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA. In certain embodiments, the RNA is mRNA.

[0082] 1.DNA The methods disclosed herein must have an RNA source. RNA can be obtained or isolated from cells or tissues. Alternatively, RNA may be prepared de novo through chemical synthesis. In various embodiments, RNA can be in vitro transcribed from a DNA source (e.g., isolated genomic DNA, plasmid DNA, or linear / linearized DNA). In various embodiments, RNA is obtained from an in vitro transcription (IVT) assay using linearized plasmid DNA. In some embodiments, RNA is obtained from an in vitro transcription (IVT) assay using linearized DNA / vector.

[0083] As used herein, the term "plasmid DNA" or "plasmid DNA vector" refers to a circular nucleic acid molecule, preferably an artificial / recombinant DNA molecule. In some embodiments, the plasmid DNA vector can be linearized. Alternatively, "linear DNA", "linear DNA vector", or "linear vector" refers to a linear nucleic acid molecule, preferably an artificial / recombinant linear DNA molecule. A plasmid or linear DNA vector in the context of the present disclosure is suitable for incorporating or carrying a desired nucleic acid sequence or gene of interest, such as a nucleic acid sequence comprising a sequence encoding an RNA and / or an open reading frame (ORF), encoding at least one polypeptide or gene of interest. Exemplary plasmids useful in the methods described herein include, but are not limited to, pUC-based vectors, e.g., pUC19. Exemplary linear DNA vectors include, but are not limited to, pJAZZ®, pSMART® (Lucigen™), and Doggybone™ / dbDNA® (Touchlight) vectors.

[0084] Expression vectors can be used to produce expression products such as RNA, e.g., mRNA, in a process called RNA in vitro transcription. For example, an expression vector can include sequences required for RNA in vitro transcription of a sequence stretch of the vector, such as a promoter sequence, e.g., an RNA promoter sequence.

[0085] Preferably, the DNA vector comprises a multiple cloning site, an RNA promoter sequence, an RNA poly(A) tail, optionally a selection marker (such as an antibiotic resistance factor), and a sequence suitable for propagation of the vector, such as an origin of replication. In certain embodiments, the DNA vector or expression vector comprises a promoter of a DNA-dependent RNA polymerase, such as T3, T7, and Sp6. The plasmid DNA may also comprise a restriction site for linearization.

[0086] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule that comprises a nucleic acid sequence that encodes an RNA sequence to be in vitro transcribed. Template DNA thus comprises all elements necessary for in vitro transcription, in particular a promoter element for binding of a DNA-dependent RNA polymerase, such as T3, T7, and SP6 RNA polymerase, operably linked 5' to the DNA sequence encoding the target RNA sequence. Template DNA may also comprise a sequence encoding a poly(A) tail located 3' to the gene of interest.

[0087] Methods for generating, replicating, and cloning the recombinant templates or plasmid DNA described herein are known in the art.

[0088] The term "template DNA" may refer to a plasmid DNA vector that includes a nucleic acid sequence that encodes an RNA sequence. Furthermore, the "template DNA" may be a linear or circular DNA molecule. In certain embodiments, the template DNA is a linearized / digested plasmid DNA molecule.

[0089] A linearized template DNA plasmid can be obtained by contacting a plasmid DNA with a restriction enzyme under suitable conditions so that the restriction enzyme cuts the plasmid DNA at its recognition site and destroys the plasmid structure. If the plasmid DNA contains only one recognition site for the restriction enzyme, the linearized template DNA has the same number of nucleotides as the plasmid DNA. If the plasmid DNA contains more than one recognition site for the restriction enzyme, the linearized template DNA has fewer nucleotides than the plasmid DNA. The linearized template DNA is then a fragment of the plasmid DNA and contains the elements required for RNA in vitro transcription, i.e., the promoter element and the template DNA element for RNA transcription. Restriction enzymes suitable for DNA cutting and / or linearization of the plasmid DNA are known in the art and include, but are not limited to, BciVI, XbaI, SpeI, HindIII, NotI, EcoRI, NdeI, BsaI, AfIII, HindIII, and SapI. In some embodiments, the restriction enzyme is a type IIS restriction enzyme. Type IIS restriction enzymes include, but are not limited to, AcuI, ALwI, BoaeI, BbsI, BbsI-HF, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspMI, MspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphhI, HpyAV, MboII, MlyI, MmeI, MnlI, NmeAIII, PaqCI, PleI, PpiI, PsrI, SapI, and SfaNI. In a particular embodiment, the restriction enzyme is BsaI.

[0090] A linear DNA vector / template may also be restricted by an endonuclease, hi some embodiments, the linear DNA vector / template is contacted with a restriction enzyme to generate a terminal adenine (A) nucleotide.

[0091] In some embodiments, following restriction, the plasmid or linear DNA template is filtered (e.g., by ultrafiltration and / or diafiltration) with a suitable solvent, such as water, TE (Tris-EDTA), Tris HCl pH 7.5, HEPES / phosphate, etc.

[0092] The linearized or linear DNA template can be purified before being used as a template for in vitro transcription. For example, the linearized or linear DNA template can be purified by phenol / chloroform extraction followed by alcohol precipitation, chromatographic or filtration methods, or silica-based DNA capture methods. This step also ensures the reduction of impurities (e.g., proteins) from previous manufacturing steps, including E. coli proteins, restriction enzymes, and BSA (contained in the reaction buffer).

[0093] In various embodiments, the process further comprises treating the sample with DNase to remove residual plasmid DNA template (circular or linear residual DNA). In some embodiments, the DNase treatment step occurs after the IVT step and / or after the capping step. In certain embodiments, the DNase is DNase I.

[0094] 2. RNA Production In certain embodiments, the linearized DNA can be used to generate RNA for use in the methods described herein using an in vitro transcription (IVT) system. An IVT system typically includes a transcription buffer, nucleotide triphosphates (NTPs), RNase inhibitors, and RNA polymerase. Methods of in vitro transcription are known in the art. See, for example, Beckert et al., Methods Mol Biol. 2011; 703: 29-41. Examples of commercially available kits for IVT include, but are not limited to, HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England BioLabs™), MEGAscript™ T7 Kit (ThermoFisher Scientific™), TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific™), Riboprobe™ or RiboMAX™ RNA Production System (Promega™), AmpliScribe™ T7 Transcription kits (Lucigen™), and RNAMaxx™ (Agilent Technologies™).

[0095] Alternatively, the IVT assay can be assembled and performed in-house by obtaining each component separately and using methods known in the art. NTPs can be manufactured in-house or purchased from commercial suppliers (e.g., Trilink® and New England BioLabs®). Any number of RNA polymerases or variants thereof can be used in the methods described herein and are readily available through commercial suppliers (e.g., New England BioLabs®, ThermoFisher Scientific™, and MilliporeSigma™). The polymerase can be selected from, but is not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerases, such as, but not limited to, polymerases that can incorporate modified nucleic acids.

[0096] A typical in vitro transcription reaction includes an RNA polymerase, such as T7 RNA polymerase, a DNA template, nucleotides (NTPs), MgCl2, and a buffer, such as HEPES or Tris. The IVT reaction may also include dithiothreitol (DTT) and / or spermidine, RNase inhibitors, pyrophosphatase, and / or EDTA. The in vitro transcription reaction may proceed, for example, at 37° C. for 4 hours under constant mixing.

[0097] 3. Capping reaction In some embodiments, the RNA used in the methods described herein is capped. Capping the RNA maximizes the efficiency of expression in cells by increasing stability and reducing degradation. In some embodiments, the RNA molecules used in the methods are synthesized in vitro by incubating uncapped RNA in the presence of a capping enzyme system. In some embodiments, the RNA is enzymatically capped at the 5' end after in vitro transcription. In some embodiments, the RNA is co-transcriptionally enzymatically capped at the 5' end. Thus, capping can be performed either before or after further purification of the RNA, such as, for example, oligo-dT purification. In some embodiments, oligo-dT affinity purification and ultrafiltration / diafiltration are performed before the capping reaction.

[0098] As used herein, the term "5' cap" or "5' cap structure" or "5' cap moiety" refers to a chemical modification incorporated at the 5' end of an mRNA. The 5' cap is involved in nuclear transport, mRNA stability, and translation.

[0099] In certain embodiments, the mRNAs contemplated herein comprise a 5' cap comprising a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule, which 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.

[0100] Illustrative examples of 5' caps suitable for use in certain embodiments of the mRNA polynucleotides contemplated herein include unmethylated 5' cap analogs, e.g., G(5')ppp(5')G, G(5')ppp(5')C, G(5')ppp(5')A, methylated 5' cap analogs, e.g., m 7 G(5')ppp(5')G,m 7 G(5')ppp(5')C, and m 7 G(5')ppp(5')A, a dimethylated 5' cap analog, e.g., m 2,7G(5')ppp(5')G,m 2,7 G(5')ppp(5')C, and m 2,7 G(5')ppp(5')A, trimethylated 5' cap analogs, e.g., m 2,2,7 G(5')ppp(5')G,m 2,2,7 G(5')ppp(5')C, and m 2,2,7 G(5')ppp(5')A, a dimethylated symmetric 5' cap analog, e.g., m 7 G(5')pppm 7 (5') G, m 7 G(5')pppm 7 (5') C and m 7 G(5')pppm 7 (5')A, as well as anti-reverse 5' cap analogs, such as anti-reverse cap analog (ARCA) caps, shown as 3'O-Me-m 7 G(5')ppp(5')G, 2'O-Me-m 7 G(5')ppp(5')G, 2'O-Me-m 7 G(5')ppp(5')C, 2'O-Me-m 7 G(5')ppp(5')A,m 7 2'd(5')ppp(5')G,m 7 2'd(5')ppp(5')C,m 7 2'd(5')ppp(5')A, 3'O-Me-m 7 G(5')ppp(5')C, 3'O-Me-m 7 G(5')ppp(5')A,m 7 3'd(5')ppp(5')G,m 7 3'd(5')ppp(5')C,m 7 3'd(5')ppp(5')A, and their tetraphosphate derivatives) (see, e.g., Jemielity et al., RNA, 9:1108-1122 (2003)).

[0101] In certain embodiments, the mRNA is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, 7G(5')ppp(5')N, where N is any nucleoside. 7 5' cap which is

[0102] In some embodiments, the mRNA comprises a 5' cap, where the cap is a cap 0 structure (the cap 0 structure lacks a 2'-O-methyl residue of the ribose attached to bases 1 and 2), a cap 1 structure (the cap 1 structure has a 2'-O-methyl residue attached to both bases 2 and 3), or a cap 2 structure (the cap 2 structure has a 2'-O-methyl residue).

[0103] For example, RNA can be enzymatically capped at the 5' end using vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to yield a Cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, using 2'O-methyltransferase with vaccinia guanylyltransferase results in a Cap 1 structure, where in addition to the Cap 0 structure, the 2'OH group is methylated on the penultimate nucleotide. S-adenosyl-L-methionine (SAM) is the cofactor utilized as the methyltransfer reagent.

[0104] In one embodiment, the mRNA is 7 Contains a G(5')ppp(5')G cap.

[0105] In one embodiment, the mRNA includes an ARCA cap or a modified ARCA cap.

[0106] In various embodiments, the RNA is co-transcriptionally capped or enzymatically capped in a separate reaction. The 5'-end cap may comprise an endogenous cap or a cap analog. The 5'-end cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0107] Further examples of 5' cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted base moiety, 3'-2'-inverted base moiety, 3'-2'-inverted base moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or a bridged or non-bridged methylphosphonate moiety. Further modified 5'-cap structures that may be used in the context of the present invention are Cap 1 (methylation of the ribose of the nucleotide adjacent to m7GpppN), Cap 2 (methylation of the ribose of the second nucleotide downstream of m7GpppN), Cap 3 (methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap 4 (methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse CAP analog, modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0108] In eukaryotes, at least three enzymatic activities are required to generate a functional Cap 0 (RNA triphosphatase (TPase), RNA guanylyltransferase (GTase), and guanine-N7 methyltransferase (guanine-N7 MTase). For the Cap 1 structure, an additional m7G-specific 2'O methyltransferase (2'O MTase) is required to methylate the +1 ribonucleotide at the 2'O position of the ribose. Eukaryotic capping enzymes are known in the art (Nucleic Acids Research, Vol. 44, No. 16, 19 Sep. 2016, pp. 7511-7526).

[0109] Viral RNA capping enzymes are also known in the art. In some cases, viral capping enzymes are known to combine enzymatic activity with multifunctional proteins. For example, flaviviruses, dengue viruses, West Nile viruses, and paramyxoviruses combine GTase and MTase activity with their RNA polymerase (RdRp). Alternatively, vaccinia virus capping enzyme and bluetongue virus capping enzyme combine all the necessary enzymatic activities of RNA capping to generate cap 0 or cap 1. Therefore, due to its simplicity and effectiveness, vaccinia virus capping enzyme, vaccinia guanylyltransferase, is often the preferred capping enzyme, but is not essential. Other viral capping enzymes known in the art include, but are not limited to, chlorella virus, alphavirus, rhabdovirus, and vesicular stomatitis virus capping enzymes. In some embodiments, polyadenylated mRNA is capped at its 5' end using vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine (SAM) to produce a cap 0 structure. In some embodiments, polyadenylated mRNA is capped at its 5' end using vaccinia guanylyltransferase, guanosine triphosphate, S-adenosyl-L-methionine (SAM), and 2'-O-methyltransferase to produce a cap 1 structure.

[0110] Capping methods and conditions are known in the art (see, e.g., Wiley Interdiscip Rev RNA, 2010 Jul-Aug;1(1):152-172, and Nat Rev Microbiol. 2011 Dec 5;10(1):51-65. An exemplary capping reaction includes S-adenosylmethionine chloride (SAM), RNase inhibitor, buffer (e.g., NEB capping buffer), GTP, vaccinia enzyme, mRNA cap 2'-O-methyltransferase, and EDTA. The reaction is carried out at 37°C under constant mixing.

[0111] 4. Affinity Chromatography To date, the primary means for purifying biological products, such as monoclonal antibodies, therapeutic proteins, vaccines, and other biological products (including DNA and RNA), has been through the use of capture chromatography, e.g., affinity chromatography. As used herein, the terms "capture chromatography" and "affinity chromatography" refer to a chromatographic component, or associated method steps, including binding and elution of a desired product (e.g., RNA) to and from a column. Capture or affinity chromatography typically uses selective non-covalent interactions between an analyte and a specific molecule (e.g., a specific ligand bound to the chromatographic medium). For example, capture or affinity chromatography may use Protein A, Protein G, an antibody (e.g., an anti-dsRNA antibody), a specific substrate / probe (e.g., oligo-dT), a ligand, or an antigen as a capture reagent. The capture reagent then migrates (links or binds) to a resin / surface within the column, and the sample passes over the resin / surface (i.e., through the column). The bound product is then eluted from the column.

[0112] Systems for chromatography (e.g., liquid chromatography) are known to those of skill in the art, such as high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UHPLC), and fast protein liquid chromatography (FPLC; e.g., AKTA™ system). Chromatography columns suitable for use in the methods described herein are also known to those of skill in the art. Columns can be of any suitable volume / size, e.g., 0.2 mL, 1.0 mL, 5.0 mL, or 10 mL. Exemplary manufacturers of chromatography columns, systems, and materials include, but are not limited to, Sigma-Aldrich™, ThermoFisher Scientific™, Waters™, Bio-Rad Laboratories, PerkinElmer®, and Cytiva.

[0113] The column may comprise a suitable resin / surface for retaining the substrate or probe. Suitable resin / surface materials are known in the art. Examples of materials that can be used as surfaces include acrylic, carbon (e.g., graphite, carbon fiber), cellulose (e.g., cellulose acetate), ceramics, controlled pore glass, cross-linked polysaccharides (e.g., agarose or Sepharose™), gels, glass (e.g., modified or functionalized glass), gold (e.g., atomically smooth Au(111)), graphite, inorganic glass, inorganic polymers, latex, metal oxides (e.g., SiO2, TiO2, stainless steel), semi-metals, metals (e.g., atomically smooth Au(111)), mica, molybdenum sulfide, nanomaterials (e.g., highly oriented pyrolytic graphite (HOPG) nanosheets), nitriles, and the like. Examples of suitable materials include, but are not limited to, polycellulose, NYLON™, optical fiber bundles, organic polymers, paper, plastics, polyacryloylmorpholide, poly(4-methylbutene), polyethylene terephthalate), poly(vinyl butyrate), polybutylene, polydimethylsiloxane (PDMS), polyethylene, polyformaldehyde, polymethacrylate, polypropylene, polysaccharides, polystyrene, poly(styrene-divinylbenzene), polyurethane, polyvinylidene difluoride (PVDF), quartz, rayon, resins, beads, rubber, semiconductor materials, silica, silicon (e.g., silicon surface oxide), sulfides, and TEFLON®.

[0114] In various embodiments, the RNA is purified by chromatographic methods using oligodeoxythymidine (dT) probes or substrates. The mechanism of purification involves hybridization of the poly(A) tail of the RNA with an oligonucleotide ligand (oligo dT) under high salt conditions. The DNA template and / or other impurities will not bind. Furthermore, RNA transcripts that do not contain a poly(A) stretch will not bind to the resin and will not form duplexes with the affinity ligand. The polyadenylated RNA can then be eluted from the resin utilizing a low ionic strength buffer or a competitive binding oligonucleotide solution. Thus, in some embodiments, the method includes contacting the RNA sample with a first or second oligo dT probe / substrate mobilized in a chromatographic column, thereby forming an oligo dT:polyadenylated RNA complex. In some embodiments, the method includes separating unbound RNA and / or contaminants from the oligo dT:polyadenylated RNA complex. In some embodiments, the method includes eluting the polyadenylated RNA from the column and retaining the eluted RNA for further purification.

[0115] In certain embodiments, the method includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA, and purifying the single-stranded polyadenylated mRNA.

[0116] In some embodiments, the method includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds to the dsRNA to form a dsRNA:antibody complex, and removing the dsRNA:antibody complex from the sample, thereby purifying the single-stranded polyadenylated RNA.

[0117] In various embodiments, the methods described herein include more than one oligo dT probe or purification step. In some embodiments, the methods described herein include two oligo dT probe or purification steps. In some embodiments, the methods include contacting a sample containing RNA and double stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds to polyadenylated RNA. In some embodiments, the polyadenylated RNA separated from the dsRNA:antibody complex using affinity chromatography is contacted with a second oligonucleotide dT probe.

[0118] In certain embodiments, the method includes contacting an RNA sample containing single-stranded polyadenylated RNA and double-stranded RNA (dsRNA) with a first oligonucleotide dT probe that binds the polyadenylated RNA and removing unbound RNA from the sample, contacting the sample with an antibody or antigen-binding fragment thereof that binds the dsRNA to form a dsRNA:antibody complex, removing the dsRNA:antibody complex from the sample, and contacting the sample with a second oligonucleotide dT probe to capture the single-stranded polyadenylated RNA, thereby purifying single-stranded polyadenylated mRNA.

[0119] In some embodiments, the first and / or second oligonucleotide dT probe is bound to a surface. In some embodiments, the first oligonucleotide dT probe is bound to a surface. In some embodiments, the second oligonucleotide dT probe is bound to a surface. In some embodiments, the oligo dT probe is bound or covalently attached to a cellulose resin. In some embodiments, a pre-packed oligo dT column is used. Pre-packed oligo dT columns for chromatography are known in the art and are commercially available, for example, POROS™ GoPure™ (ThermoFisher Scientific).

[0120] The oligo dT substrates / probes may be of different lengths, for example, may contain about 15 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 16 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 17 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 18 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 19 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 20 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 21 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 22 to about 30 thymidine residues. In some embodiments, the oligo dT substrates / probes contain about 23 to about 30 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 24 to about 30 thymidine residues.In some embodiments, the oligo dT substrate / probe comprises about 25 to about 30 thymidine residues.

[0121] In some embodiments, the oligo dT substrate / probe comprises about 15 to about 29 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 28 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 27 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 26 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 25 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 24 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 23 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 22 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 21 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 15 to about 20 thymidine residues.

[0122] In some embodiments, the oligo dT substrate / probe comprises about 21 to about 29 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 22 to about 28 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 23 to about 27 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 24 to about 26 thymidine residues.

[0123] Oligo dT substrates / probes can be of different lengths and can contain, for example, at least about 15 thymidine residues, at least about 16 thymidine residues, at least about 17 thymidine residues, at least about 18 thymidine residues, at least about 19 thymidine residues, at least about 20 thymidine residues, at least about 21 thymidine residues, at least about 22 thymidine residues, at least about 23 thymidine residues, at least about 24 thymidine residues, at least about 25 thymidine residues, at least about 26 thymidine residues, at least about 27 thymidine residues, at least about 28 thymidine residues, at least about 29 thymidine residues, or at least about 30 thymidine residues.

[0124] In some embodiments, the oligo dT substrate / probe comprises about 15 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 16 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 17 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 18 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 19 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 20 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 21 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 22 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 23 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 24 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 25 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 26 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 27 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 28 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 29 thymidine residues. In some embodiments, the oligo dT substrate / probe comprises about 30 thymidine residues. In preferred embodiments, the oligo dT substrate / probe comprises 23 or 25 thymidine residues (SEQ ID NO: 16 and 15, respectively). In some embodiments, the oligo dT substrate / probe comprises 23 thymidine residues (SEQ ID NO: 16). In some embodiments, the oligo dT substrate / probe comprises 25 thymidine residues (SEQ ID NO: 15).

[0125] In various embodiments, the methods described herein include an additional chromatographic step to further remove dsRNA from the RNA preparation. In some embodiments, the methods include contacting a sample containing single-stranded RNA (e.g., capped polyadenylated mRNA) and dsRNA with an antibody or antigen-binding fragment thereof that binds to dsRNA, and separating the dsRNA:antibody complex from the single-stranded RNA (e.g., capped polyadenylated mRNA).

[0126] In various embodiments, the dsRNA:antibody complex is separated from the single stranded RNA by affinity chromatography. In some embodiments, the affinity chromatography comprises a 1 ml column. In some embodiments, the affinity chromatography comprises a 5 ml column. In some embodiments, the affinity chromatography comprises a 10 ml column.

[0127] Any resin / column that can bind anti-dsRNA antibodies can be used in the methods described herein to deplete antibody:dsRNA complexes and release antibodies from RNA samples. For example, Protein A or Protein G binding resins are most typically used to capture antibodies and antibody complexes. Protein A and Protein G are immunoglobin binding proteins originally isolated from bacteria. In a preferred embodiment, the resin is a Protein A binding resin or bead. Protein A resins or beads are known in the art, for example, MabCapture™ A Select ProA™ resin is commercially available.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof that binds to dsRNA is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), and single domain antibody (sdAb, camelid VHH, nanobody). In some embodiments, the antibody or antigen-binding fragment thereof that binds to dsRNA is a monoclonal antibody. In some embodiments, the antibody is selected from the group consisting of J2, J5, K1, K2, 1D3, CABT-B212, and 9D5. In a particular embodiment, the antibody is J2.

[0129] Antibodies that bind to dsRNA are known and commercially available. Commercial entities that sell one or more of the above-listed dsRNA antibodies include, but are not limited to, MilliporeSigma, Jena Bioscience, Scicons, Thermo Fisher Scientific, Absolute Antibody, and Creative Diagnostics®.

[0130] In various embodiments, the RNA sample is contacted with at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%, at least about 5.5 mol%, at least about 6 mol%, at least about 6.5 mol%, at least about 7 mol%, at least about 7.5 mol%, at least about 15 mol%, at least about 30 mol%, or at least about 60 mol% of the antibody compared to the total moles of RNA in the sample. For example, the moles of RNA can be determined by dividing the total mass of RNA in the sample by the molecular weight (MW) of the RNA transcript. The molecular weight of the RNA transcript can be determined by multiplying its predicted length by 330 g / mol (average MW of RNA nucleotides). The RNA concentration can be determined by UV absorbance at 260 nm, which can then be multiplied by the volume to obtain the total mass of RNA in the sample. The moles of anti-dsRNA antibody can be determined similarly. If the concentration of anti-dsRNA antibody is unknown, it can be determined by UV absorbance at 280 nm. If the concentration and volume of anti-dsRNA antibody are known, the total mass can simply be divided by the MW of the antibody to obtain the total moles of antibody. Once the total moles of RNA and moles of anti-dsRNA antibody are determined, the appropriate percentage of anti-dsRNA antibody can be added to the RNA sample (e.g., 7.5 mol%, 15 mol%, 30 mol%, or 60 mol% of anti-dsRNA antibody). For example, if there are 100 moles of RNA in a sample, 60 moles of anti-dsRNA antibody can be added to the RNA sample to obtain 60 mole% of anti-dsRNA.

[0131] In some embodiments, the RNA sample is contacted with at least about 1.5 mol%, at least about 7.5% mol%, at least about 15% mol%, at least about 30% mol%, or at least about 60% mol% of the antibody relative to the total moles of RNA in the sample. In certain embodiments, the RNA sample is contacted with at least about 7.5 mol% of the antibody relative to the total moles of RNA in the sample.

[0132] In various embodiments, the RNA sample is contacted with about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 15 mol%, about 30 mol%, or about 60 mol% of the antibody compared to the total moles of RNA in the sample. In certain embodiments, the RNA sample is contacted with about 7.5 mol% of the antibody compared to the total moles of RNA in the sample. In certain embodiments, the RNA sample is contacted with about 15 mol% of the antibody compared to the total moles of RNA in the sample. In certain embodiments, the RNA sample is contacted with about 30 mol% of the antibody compared to the total moles of RNA in the sample. In certain embodiments, the RNA sample is contacted with about 60 mol% of the antibody compared to the total moles of RNA in the sample.

[0133] In various embodiments, the RNA sample is contacted with about 1.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 2 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 2.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 3 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 3.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 4 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 4.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 5.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 6 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 6.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 7 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 7.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 15 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 30 mol % to about 60 mol % of the antibody compared to the total moles of RNA in the sample.

[0134] In various embodiments, the RNA sample is contacted with about 1.5 mol% to about 30 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 15 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 7.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 7 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 6.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 6 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 5.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 4.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 4 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 3.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 3 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 2.5 mol% of the antibody compared to the total moles of RNA in the sample. In some embodiments, the RNA sample is contacted with about 1.5 mol% to about 2 mol% of the antibody compared to the total moles of RNA in the sample.

[0135] 5. Filtration Impurities from the RNA preparation can be filtered out during one or more steps of the methods described herein. For example, the RNA preparation can be passed through a membrane (e.g., an ultrafiltration membrane) to remove unwanted proteins (e.g., enzymes / proteins) from previous reactions and / or to increase the RNA concentration in the preparation.

[0136] As used herein, the term "ultrafiltration" or "UF" refers to any technique in which a solution or suspension is subjected to a semipermeable membrane that retains macromolecules while allowing solvents and small solute molecules to pass through. The terms "ultrafiltration membrane" and "UF membrane" refer to a membrane having a pore size ranging from about 10 nanometers to about 100 nanometers (i.e., about 0.01 micrometers to about 0.1 micrometers). Ultrafiltration can be used to increase the concentration of RNA in a sample and / or remove impurities (e.g., proteins). RNA ultrafiltration techniques and methods are known in the art (see, e.g., Fernandez et al., "Cross flow filtration of RNA extracts by hollow fiber membrane," Acta Biotechnol., 12:49-56, 1992).

[0137] Alternatively, diafiltration can be used to perform buffer exchange and / or concentrate RNA preparations. In general, diafiltration is a technique that uses a membrane to remove, replace, or reduce the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. Thus, as used herein, the term "diafiltration" or "DF" refers to a special type of filtration in which the retentate is diluted with a solvent and refiltered to reduce the concentration of soluble permeate components. The term "retentate" refers to the portion of the sample / preparation or feed feed that is retained by the membrane, and the retentate is a stream that is concentrated with the retained species.

[0138] For example, in continuous diafiltration, solvent is added continuously to the retentate at the same rate that filtrate is produced. In this case, the retentate volume and the concentration of the retained components do not change during the process. On the other hand, in discontinuous or continuous dilution diafiltration, following the step of ultrafiltration, solvent is added to the retentate side, and if the volume of solvent added to the retentate side is not equal to or greater than the volume of filtrate produced, the concentration of the retained components will have a high concentration.

[0139] Diafiltration can be used to change the pH, ionic strength, salt composition, buffer composition, or other properties of a solution or suspension of macromolecules.

[0140] As used herein, the term "ultrafiltration / diafiltration" or "UF / DF" refers to any process, technique, or combination of techniques that achieves ultrafiltration and / or diafiltration, either sequentially or simultaneously. UF / DF techniques, methods, and membranes are known in the art. See, e.g., Eon-Duval et al., Anal Biochem. 2003 May 1;316(1):66-73.

[0141] In various embodiments, the ultrafiltration, diafiltration, and / or UF / DF processes utilize tangential flow filtration (e.g., tangential flow ultrafiltration / diafiltration). Tangential flow filtration (TFF) is a process that uses membranes to separate components in a liquid solution or suspension (e.g., a feed sample) based on size, molecular weight, or other differences. In these processes, the feed sample is pumped tangentially along the membrane surface, and particles or molecules that are too large to pass through the membrane are retained and returned to the process tank for additional passes across the membrane (i.e., recirculation) until the feed sample is sufficiently clarified, concentrated, or purified. The cross-flow nature of TFF minimizes membrane fouling and allows for large volume processing per batch.

[0142] Membranes suitable for ultrafiltration and / or diafiltration can be made of a variety of different substrates or polymers known in the art. For example, in some embodiments, the TFF cassette or hollow fiber cartridge contains membranes made of polysulfone, polyethersulfone, poly(methyl methacrylate), polyvinylidene fluoride, modified cellulose, regenerated cellulose, delta regenerated cellulose, cellulose acetate, and / or other polymers or substrates known to those skilled in the art. In some embodiments, the membrane is a polysulfone membrane. In some embodiments, the membrane is a polyethersulfone membrane. In some embodiments, the membrane is a poly(methyl methacrylate) membrane. In some embodiments, the membrane is a polyvinylidene fluoride membrane. In some embodiments, the membrane is a modified cellulose membrane. In some embodiments, the membrane is a regenerated cellulose membrane. In some embodiments, the membrane is a delta regenerated cellulose membrane. In some embodiments, the membrane is a cellulose acetate membrane. In a preferred embodiment, the membrane is a hollow fiber membrane.

[0143] Exemplary TFF cassettes / membranes useful for the methods contemplated in certain embodiments herein include, but are not limited to, TFF cassettes provided by MilliporeSigma Corporation (Burlington, Mass.), Pall Corporation (Port Washington, NY), GE Healthcare Bio-Sciences (Piscataway, NJ), and Sartorius AG (Bohemia, NY). Exemplary MilliporeSigma Corporation TFF cassettes include, but are not limited to, Pellicon® cassettes (e.g., Pellicon® 2 cassettes, Pellicon® 2 mini cassettes, Pellicon® 2 Maxi cassettes, Pellicon® 3 cassettes) with Biomax® membranes, Ultracel® membranes, or Durapore® membranes. Examples of Pall Corporation TFF cassettes include, but are not limited to, Centrasette® cassettes and Cadence® single-use cassettes. Exemplary GE Healthcare Bio-Sciences TFF cassettes include, but are not limited to, Kvick™ flow cassettes. Exemplary Sartorius AG cassettes include, but are not limited to, Hydrosart® cassettes.

[0144] In various embodiments, the methods contemplated herein include an additional step of filtration. In some embodiments, the step of filtration includes a final filter (i.e., the last filter in the method). In some embodiments, the filter is a sterile filter. In some embodiments, the filter includes a microfiltration membrane. In some embodiments, the filter includes an ultrafiltration membrane. In some embodiments, the filter includes a nanofiltration membrane. In some embodiments, the filter is a 0.22 μm filter.

[0145] D. RNA and Related Therapeutic Genes and Proteins Ribonucleic acid (RNA) is a nucleic acid molecule, i.e., a polymer made up of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and cytidine monophosphate (CMP) monomers or their analogs, which are connected to each other via a molecular backbone. The backbone is formed by phosphodiester bonds between the sugar moieties, i.e., ribose, of each nucleotide monomer (base).

[0146] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases and a wide variety of modified bases recognized in the art. Such bases are typically located at the 1' position of the nucleotide sugar moiety. Nucleotides generally include a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., deoxyribose is a sugar lacking the hydroxyl group present in ribose. Examples of natural nitrogenous bases include the purines adenosine (A) and guanidine (G), and the pyrimidines cytidine (C) and thymidine (T) (or uracil (U) in the case of RNA). The C-1 atom of deoxyribose is linked to the N-1 of a pyrimidine or the N-9 of a purine. Nucleotides are usually mono-, di-, or triphosphates. Nucleotides may be unmodified or modified at the sugar, phosphate, and / or base moieties (interchangeably referred to as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides, see, for example, WO92 / 07065 and WO93 / 15187). Examples of modified nucleobases are summarized in Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

[0147] Nucleotides may also be considered as phosphate esters of nucleosides, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, as well as modified bases, which are well known. Such bases are typically located at the 1' position of the nucleoside sugar moiety. Nucleosides generally include a base and a sugar. Nucleosides may be unmodified or modified at the sugar and / or base moieties (interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). Also, as mentioned above, examples of modified nucleobases are summarized in Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

[0148] Messenger RNA (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene. mRNA can be obtained, for example, in a cell, by transcription of a DNA sequence. In a cell, transcription of DNA typically results in the production of a premature mRNA, which is then processed into a mature mRNA. Processing of the premature RNA into a mature messenger RNA usually involves splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. Alternatively, mRNA can be transcribed from recombinant DNA, either in vitro (as described above) or in vivo. In this case, the translated recombinant DNA sequence typically does not contain introns, so no splicing of exons is required during processing.

[0149] A mature mRNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a particular peptide or protein. Typically, a mature mRNA includes a 5'-cap, an optional 5'UTR, an open reading frame, an optional 3'UTR, and a poly(A) sequence.

[0150] RNA (e.g., mRNA) useful in the methods / processes provided herein may be the product of DNA transcription (e.g., an RNA transcript) or may be chemically synthesized. An RNA transcript (e.g., an in vitro transcribed mRNA) is the polynucleotide product of an in vitro transcription reaction.

[0151] As used herein, "RNA transcript" refers to a ribonucleic acid produced by an in vitro transcription reaction using a DNA template and an RNA polymerase. The RNA transcript typically includes the coding sequence of a gene of interest and a poly(A) tail. The RNA transcript may include modifications, such as modified nucleotides. As used herein, the term RNA transcript includes and is interchangeable with mRNA, whether transcribed from a DNA template or chemically synthesized.

[0152] RNA transcripts and mRNA are typically single-stranded (ssRNA), while double-stranded RNA (dsRNA) is a common by-product of transcription (e.g., in vitro transcription). It is hypothesized that dsRNA can be generated in multiple ways, including, but not limited to, turnaround transcription (cis), random priming of abortive transcripts (cis / trans), and / or antisense transcription of DNA. Regardless of the mechanism of dsRNA formation, dsRNA is typically known to be toxic to cells; for example, when dsRNA is administered in vivo, recipient cells may perceive it as an invading virus that can induce an immune response.

[0153] The RNA transcripts (e.g., mRNA samples) purified in the methods / processes described herein are not limited by the source of RNA. In some embodiments, the RNA is synthesized by in vitro transcription of a DNA template that includes a gene cloned into a linearized or linearized plasmid vector, or by in vitro transcription of a DNA template that is synthesized by PCR or RT-PCR (i.e., by IVT of a PCR amplification product). The RNA can be capped as described above. In one embodiment, the RNA transcript includes a 5' cap that is typically added post-transcriptionally.

[0154] In certain embodiments, the RNA is polyadenylated (poly(A)). Poly(A) may be encoded in the DNA template or added post-transcriptionally. In certain embodiments, the RNA contemplated herein comprises a poly(A) tail to help protect the RNA from exonuclease degradation, stabilize the RNA, and facilitate translation. In certain embodiments, the RNA comprises a 3' poly(A) tail structure. Methods for polyadenylation of RNA are known in the art (PL Wigley et al. Mol Cell Biol. 1990 Apr;10(4):1705-1713, and Wakiyama et al., Biochimie. 1997 Dec;79(12):781-5).

[0155] In certain embodiments, the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides, or any intervening number of adenine nucleotides. , 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, or 275 or more adenine nucleotides.

[0156] In certain embodiments, the length of the poly(A) tail is from about 10 to about 500 adenine nucleotides, from about 50 to about 500 adenine nucleotides, from about 100 to about 500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 200 to about 500 adenine nucleotides, from about 250 to about 500 adenine nucleotides, from about 300 to about 500 adenine nucleotides, from about 50 to about 450 adenine nucleotides, from about 50 to about 500 adenine nucleotides, from about 60 to about 650 adenine nucleotides, from about 70 to about 750 adenine nucleotides, from about 80 to about 850 adenine nucleotides, from about 90 to about 950 adenine nucleotides, from about 100 to about 500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 200 to about 500 adenine nucleotides, from about 250 to about 500 adenine nucleotides, from about 300 to about 500 adenine nucleotides, from about 50 to about 450 adenine nucleotides, from about 50 to about 500 adenine nucleotides, from about 60 to about 750 adenine nucleotides, from about 70 to about 850 adenine nucleotides, from about 80 to about 950 adenine nucleotides, from about 90 to about 1000 adenine nucleotides, from about 100 to about 1500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 100 to about 1500 aden adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 400 adenine nucleotides nucleotides, about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides, or about 250 to about 300 adenine nucleotides, or any intervening range of adenine nucleotides.

[0157] In some embodiments, the RNA transcript comprises a 5'UTR and a 3'UTR.

[0158] Terms describing the orientation of a polynucleotide (e.g., an RNA transcript or mRNA) include 5' (usually the end of the polynucleotide having a free phosphate group) and 3' (usually the end of the polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus", or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus", or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation, or a 3' to 5' sequence written in a 5' to 3' orientation.

[0159] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTAC 5'. The latter sequence is often written as the reverse complement, 5'CATGACT 3', with the 5' end on the left and the 3' end on the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules, or there can be "complete" or "total" complementarity between the nucleic acids.

[0160] Furthermore, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can code for fragments of the polypeptides or variants thereof contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon preferences, are specifically contemplated in certain embodiments. In one embodiment, a polynucleotide is provided that comprises a specific allele sequence. An allele is an endogenous polynucleotide sequence that is modified as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides.

[0161] The RNA transcript may be a coding RNA (e.g., mRNA) that encodes a protein or a fragment or variant thereof, including, but not limited to, a secreted protein, a plasma membrane protein, a cytoplasmic or cytoskeletal protein, an intracellular membrane-associated protein, a protein associated with a human disease, a targeting moiety, a fusion protein, an enzyme, an endonuclease, an exonuclease, a CRISPR-associated nuclease (e.g., Cas9 and its variants), a meganuclease or a homing endonuclease (HE), a transcription activator-like effector nuclease (TALEN), a megaTAL, a zinc finger nuclease, a tumor antigen, a pathogenic antigen, an allergenic antigen, an autoimmune antigen, or a protein encoded by the human genome. RNA sequences that encode peptides or proteins can be easily identified by those skilled in the art by using public and private databases, such as NCBI GenBank or PubMed. In certain embodiments, the coding RNA may be, for example, an mRNA, a viral RNA, or a replicon RNA.

[0162] In various embodiments, the RNA transcript or mRNA encodes a nuclease (e.g., an endonuclease or an exonuclease). The term "endonuclease" refers to an enzyme that cleaves phosphodiester bonds in a polynucleotide chain. The polynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (e.g., containing bases other than A, C, G, and T). Endonucleases may cleave polynucleotides symmetrically to leave "blunt" ends or to form overhangs that may be referred to as "sticky ends" at positions that are not directly opposed. The methods and compositions described herein may be applied to cleavage sites generated by endonucleases. Endonucleases include, but are not limited to, gene editing enzymes such as meganucleases, homing endonucleases (HEs), megaTALs, TALENs, zinc finger nucleases, CRISPR-associated nucleases, or functional variants thereof.

[0163] In various embodiments, the RNA transcript or mRNA encodes a gene editing endonuclease. In some embodiments, the gene editing endonuclease is a meganuclease, a homing endonuclease (HE), a megaTAL, a TALEN, a zinc finger nuclease, or a CRISPR-associated nuclease (e.g., Cas9). In certain embodiments, the gene editing endonuclease is a meganuclease, a homing endonuclease (HE), or a megaTAL.

[0164] The terms "homing endonucleases" and "meganucleases" are used interchangeably and refer to naturally occurring nucleases that recognize cleavage sites (e.g., target sites) of 12-45 base pairs and are generally grouped into five families based on sequence and structural motifs: LAGLIDADG (SEQ ID NO: 14), GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. See, e.g., Stoddard Structure. 2011 Jan 12;19(1):7-15.

[0165] "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence within a target gene. See, e.g., Boissel et al. Methods Mol Biol (2015); 1239: 171-96. In some embodiments, a megaTAL further comprises one or more linkers and / or additional functional domains, such as a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, a 3'-5' exonuclease (e.g., Trex2, ExoI, or ExoX), a 5' flap endonuclease, a helicase, or an end-processing enzyme domain of an end-processing enzyme exhibiting template-independent DNA polymerase activity.

[0166] The term "clustered regularly interspaced short palindromic repeats" or "CRISPR" refers to a family of DNA sequences originally found in the genomes of prokaryotes such as bacteria and archaea, and are used to detect and destroy DNA from bacteriophages. CRISPR sequences in combination with nucleases (e.g., Cas nucleases, CRISPR-Cas) can also be used to edit genes in organisms and have a variety of applications in research, gene editing, and therapy. See, for example, Nature Biotechnology volume 38, pages 824-844 (2020).

[0167] The terms "CRISPR-associated nuclease" and "Cas nuclease" are used interchangeably and refer to an RNA-guided sequence-specific nuclease that uses a CRISPR sequence as a guide to generate specific single- or double-stranded breaks in DNA. In general, targeting by the CRISPR-Cas system requires a short sequence known as a protospacer adjacent motif (PAM) to occur near the target DNA site.

[0168] The term "zinc finger nuclease" (ZFN) refers to an artificial restriction enzyme generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to bind to a desired target site. In some embodiments, the cleavage domain comprises the non-specific cleavage domain of FokI. In other embodiments, the cleavage domain comprises all or an active portion of another nuclease.

[0169] The term "TAL effector nuclease" (TALEN) refers to a nuclease that comprises a TAL effector domain (TALE) fused with a nuclease domain. TAL effector DNA binding domains isolated from the plant pathogen Xanthomonas have been described (see Boch et al., (2009) Science 29 Oct. 2009 (10.1126 / science.117881), and Moscou and Bogdanove, (2009) Science 29 Oct. 2009 (10.1126 / science.1178817)). These DNA binding domains can be engineered to bind to desired targets and fused with nuclease domains, such as FokI nuclease domains, to induce TAL effector domain-nuclease fusion proteins.

[0170] A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule binds and / or cleaves, provided that sufficient conditions for binding and / or cleavage exist. When referring to a polynucleotide sequence or SEQ ID NO: that references only one strand of a target site or target sequence, it is understood that the target site or target sequence bound and / or cleaved by a nuclease variant is double-stranded and includes the reference sequence and its complementary strand. In various embodiments, the target site is within an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of gamma-globin gene expression and / or HbF, or an immunosuppressive signaling gene.

[0171] In various embodiments, the nuclease (e.g., endonuclease, HE, megaTAL, TALEN, ZFN, or CRISPR-Cas) target site is within an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of gamma-globin gene expression and HbF, or an immunosuppressive signaling gene. In some embodiments, the target site is within a gene selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes.

[0172] In some embodiments, the target site is a sequence in the human TRAC gene. In some embodiments, the target site is a sequence in the PD1 gene. In some embodiments, the target site is a sequence in the PCSK9 gene. In some embodiments, the target site is a sequence in BCL11A. In some embodiments, the target site is a sequence in BCL11A.

[0173] Other target genes include α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD1, alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, and CD79. b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY) , L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid;These may include, but are not limited to, placenta specific 1 (PLAC1), predominantly expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1) gene, and Wiskott-Aldrich syndrome (WAS) gene;

[0174] In some embodiments, the nuclease (e.g., endonuclease, HE, megaTAL, TALEN, ZFN, or CRISPR-Cas) target site is within a gene selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T-lymphocyte antigen-4 (CTLA-4), band T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig inhibitor of T-cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), IL10Rα, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes.

[0175] In various embodiments, the nuclease (e.g., endonuclease, HE, megaTAL, TALEN, ZFN, or CRISPR-Cas) target site is within the TRAC (TCRα) gene, the PDCD1 (PD-1) gene, or the PCSK9 gene. In certain embodiments, the TCRα megaTAL RNA comprises the sequence set forth in SEQ ID NO: 2 or 3 (see, e.g., WO2018 / 071565, which is incorporated by reference herein in its entirety). In certain embodiments, the PD-1 megaTAL RNA comprises the sequence set forth in SEQ ID NO: 5 or 6 (see, e.g., WO2018 / 049226, which is incorporated by reference herein in its entirety). In certain embodiments, the PCSK9 megaTAL RNA comprises the sequence set forth in SEQ ID NO: 8 or 9 (see, e.g., WO2019 / 070974, which is incorporated by reference herein in its entirety).

[0176] In various embodiments, the RNA transcript encodes an exonuclease, a terminal processing enzyme, or a fragment or variant thereof. In some embodiments, the RNA transcript encodes an exonuclease, a terminal processing enzyme, or a fragment or variant thereof, such as Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, ExoI, ExoT, ExoIII, ExoX, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, N The exonuclease, terminal processing enzyme, or fragment or variant thereof selected from the group consisting of DK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4, and UL-12. In some embodiments, the exonuclease is Trex2 or a biologically active fragment thereof. In certain embodiments, the Trex2 RNA comprises the sequence set forth in SEQ ID NO: 11 or 12.

[0177] In various embodiments, the RNA transcript may encode a disease-associated protein or polypeptide (e.g., a therapeutically active protein or polypeptide). In some embodiments, the therapeutically active protein or polypeptide is α-globin, β-globin, γ-globin, FVIII, or antihemophilic factor (AHF), ATP-binding cassette subfamily D member 1 (ABCD1), adenosine deaminase, interleukin 2 receptor gamma, tripeptidyl peptidase 1, alpha-L iduronidase, or iduronate 2-sulfatase.

[0178] Alternatively, the selected RNA sequence may be any RNA as defined herein, in particular messenger RNA (mRNA), small interfering RNA (siRNA), antisense RNA, CRISPR RNA, circular RNA (circRNA), ribozyme, aptamer, riboswitch, immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) small nucleolar RNA (snoRNA), microRNA (miRNA), or Piwi-interacting RNA (piRNA). In some embodiments, the RNA may comprise naturally occurring and / or modified nucleotides.

[0179] In one embodiment, the RNA (e.g., mRNA) is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, 5-hydroxy ... Auridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2 -thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine cytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-Hydroxyisopentenyl)adenosine, 2-Methylthio-N6-(cis-Hydroxyisopentenyl)adenosine, N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-adenine, 2-Methoxy-adenine, Inosine, 1-Methyl-inosine, Wyosine, Wyobutosine, 7-Deaza-guanosine, 7- The modified nucleosides include one or more modified nucleosides selected from the group consisting of deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0180] In one embodiment, the RNA (e.g., mRNA) is selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio -uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0181] In one embodiment, the RNA (e.g., mRNA) is selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, The modified nucleoside comprises one or more modified nucleosides selected from the group consisting of 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0182] In one embodiment, the RNA (e.g., mRNA) is selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopurine), ... The modified nucleosides include one or more modified nucleosides selected from the group consisting of N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0183] In one embodiment, the RNA (e.g., mRNA) comprises one or more modified nucleosides selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0184] In one embodiment, the RNA (e.g., mRNA) comprises one or more pseudouridines, one or more 5-methyl-cytosines, and / or one or more 5-methyl-cytidines. In one embodiment, the mRNA comprises one or more pseudouridines. In one embodiment, the mRNA comprises one or more 5-methyl-cytidines. In one embodiment, the mRNA comprises one or more 5-methyl-cytosines. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

[0185] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0186] The foregoing embodiments have been described in detail with reference to figures and examples for purposes of clarity and understanding, but in light of the teachings contemplated herein, it will be readily apparent to those skilled in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0187] Example 1 A new method for removing dsRNA from therapeutic RNA preparations We have developed a new and improved method / process for removing dsRNA that is surprisingly effective and reduces cytotoxicity in vivo. Figures 1A-1F show exemplary method unit operations. Briefly, the process uses unamplified linear DNA (e.g., from digested plasmids, etc.) as a template encoding a gene of interest. An in vitro transcription reaction is used to synthesize RNA transcripts from the linear DNA (e.g., using T7 phage polymerase and nucleotide triphosphates). The RNA transcripts are post-translationally (or co-transcriptionally) enzymatically capped at the 5' end using a capping enzyme, e.g., vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine, resulting in a Cap 0 structure. Alternatively, a 2'O-methyltransferase can be used to obtain a Cap 1 structure. The Cap 1 structure contains a methylated 2'OH group at the penultimate nucleotide. In some embodiments, capping is performed co-transcriptionally using known methods and / or commercially available products (e.g., CleanCap®).

[0188] Then, the antibody that binds dsRNA is batch-incubated with the RNA transcripts to bind dsRNA impurities. The sample is depleted of antibody-dsRNA complexes and free antibody using a resin (e.g., MabCapture™ A Select ProA™ resin) to release the antibody. The RNA transcripts are also chromatographically purified during the reaction unit operation by affinity chromatography using an oligo dT affinity column / resin (e.g., an oligo dT cellulose resin / column or a POROS™ Oligo(dT)25 column (SEQ ID NO: 15)) and diafiltered into the desired formulation buffer. The mRNA is filtered through a 0.22 μm filter as a final step.

[0189] Example 2 J2 antibody and oligo(dT) affinity chromatography effectively remove dsRNA from RNA preparations TCRa megaTAL mRNA (SEQ ID NO:2) was purified using the process unit operations described in Example 1 (see also FIG. 1C). Specifically, dsRNA was removed from the RNA sample using anti-dsRNA antibody J2 coupled to MabCapture™ A Select ProA™ resin (ThermoFisher Scientific™). The use of 1 mL and 5 ml columns of ProA resin / J2 antibody ("1xProA column" and "5xProA column"), as well as a 5 ml column (ProA resin / J2 antibody) with an additional step of affinity chromatography purification (oligo dT) after the step of J2 antibody purification ("5xProA column+dT"), was tested. dsRNA content was measured by dsRNA dot blot assay as described in Kariko et al., Nucleic Acids Res. 2011 Nov;39(21):e142. Briefly, mRNA lots were blotted onto charged Nytran™ membranes along with synthetic 100% double-stranded RNA controls. The membranes were dried, blocked, and incubated overnight with J2 anti-dsRNA IgG2a monoclonal antibody. After several washes, fluorescent secondary antibodies were used to bind the J2 antibody. After several washes, images were captured using a LI-COR Odyssey CLx Imaging System, and analysis of the proportion of double-stranded RNA in the mRNA lots was performed by fluorescence intensity comparison compared to the 100% control.

[0190] In the 1 mL column, there was a significant depletion of dsRNA content, as indicated by the decrease in fluorescence (Figures 2A and 2B). Further depletion of antibody:dsRNA complexes was observed by increasing the volume of ProA resin, i.e., by using a 5 mL column. Surprisingly, however, further depletion of antibody:dsRNA complexes was achieved by adding an affinity chromatography purification step (i.e., an oligo dT purification step) after the step of J2 antibody purification (Figure 2B). Furthermore, direct dot blots of secondary fluorescently labeled samples show that the residual fluorescent signal detected in the dot blot in Figure 2B is due to residual J2 antibody that has passed through the ProA column (Figure 2C). Thus, the assessment of dsRNA content by dsRNA dot blot indicates that when combining the above-mentioned antibody and oligo dT-based purifications, the proportion of dsRNA in the mRNA sample is not detectable by the assay.

[0191] Example 3 J2 antibody titration J2 titration experiments were performed to determine the effective amount of J2 mAb for double-stranded RNA (dsRNA) clearance. PD1 megaTAL (SEQ ID NO:5, approx. 3000 nt) and Trex2 (SEQ ID NO:11, approx. 1000 nt) mRNAs were prepared using an in-house mRNA production process. Briefly, mRNA material was generated by in vitro transcription and capped at the 5' end with a Cap 0 structure prior to J2 titration experiments. The amount of J2 was calculated according to the mRNA molecules (up to 60 mol%). Samples were incubated with an appropriate amount of J2 for 30 min at room temperature, followed by addition of ProA resin to capture the antibody:dsRNA complex (incubated at room temperature for 1 h). The purified mRNA material was then collected through a vacuum manifold. J2 dot blots (as above) and impedance assays (described below) were performed to determine dsRNA content and toxicity to BJ fibroblasts.

[0192] The cytotoxicity of the mRNA lots was evaluated via cell impedance assay using BJ fibroblasts and the xCELLigence™ RTCA MP instrument from ACEA Biosciences, Inc. The xCELLigence instrument uses non-invasive electrical impedance monitoring to continuously measure cell viability in the form of a "cell index" value. Cells were attached to ACEA's E-plates containing interdigitated electrodes and grown for 24 hours. Cells were then transfected with the mRNA lots and the double-stranded mRNA killing control and monitored for 72 hours post-transfection. The ACEA software was used to analyze the cell index value of each well over a 72-hour post-transfection window to report the value of the cell index slope. The cell index slope of a given mRNA lot was compared to the cell index slope of the LNP-only and double-stranded killing control to obtain an index of cytotoxicity.

[0193] Titration experiments with both mRNA constructs demonstrated effective dsRNA clearance at ≥7.5 mol% J2 (Figures 3A and 3B). Furthermore, in vitro toxicity results correlated closely with dsRNA content, showing decreased cytotoxicity at lower dsRNA levels (Figures 3C and 3D).

[0194] Example 4 In vivo gene editing and mRNA characterization Crude in vitro transcribed (IVT) RNA material of PCSK9 megaTAL and Trex2 mRNA (SEQ ID NOs: 8 and 11, respectively) generated in-house was sent to a commercial vendor for capping and purification by either silica resin (commercial-silica) or HPLC (commercial-HPLC). The same crude IVT material was purified in-house using poly(A) mRNA isolation (oligo dT purification) and dsRNA depletion (J2 purification) as described above. PCSK9 megaTAL mRNA purified using the three methods was compared in three separate in vitro assays (Figures 4A-4F). mRNA length was measured by running the mRNA on an Advanced Analytical, capillary electrophoresis-based fragment analyzer using their standard RNA analysis reagents according to the manufacturer's recommended protocol. Area under the curve was measured using ProSize software (Agilent Technologies, Inc) and the average total area of ​​selected peaks for three replicates was plotted (Figure 4A).

[0195] Double-stranded mRNA (dsRNA) can be toxic when delivered in vivo. To measure the amount of dsRNA in the mRNA preparation, a dsRNA dot blot assay was performed as described above. The J2 / dT mRNA production process produces mRNA with undetectable levels of dsRNA, which is comparable or better than HPLC- and silica-purified mRNA (Figure 4B).

[0196] mRNA toxicity was measured in an in vitro cell proliferation assay using the RTCA iCELLigence™ impedance-based assay from ACEA Biosciences, Inc. Human BJ fibroblast (ATCC, CRL-2522) cells were seeded onto iCELLigence plates and allowed to adhere for 18-24 hours. mRNA was formulated into Lipofectamine MessengerMax transfection reagent according to the manufacturer's recommended protocol and used to transfect the cells. The amount of cell proliferation was measured for 48 hours, and the slope of proliferation was graphed as an index of toxicity (Figure 4C).

[0197] PCSK9 megaTAL and Trex2 mRNA purified using the three methods were formulated in liquid nanoparticles (LNPs) (Acuitas Therapeutics) at a 1.0:1.0 molar ratio. The mRNA / LNP formulations were diluted in phosphate-buffered saline (PBS) and administered at a dose of 1 mg / kg to five Balb / C mice per condition (via tail vein injection) (Figures 4D-4F). INDEL analysis was performed using next-generation amplicon sequencing and graphed as fold change compared to the silica condition (Figure 4D).

[0198] The relative toxicity of each mRNA preparation was assayed by taking mandibular bleeds from animals 24 hours after administration and measuring aspartate aminotransferase (AST) enzyme levels (Figure 4E). The in vivo immunogenicity of the purified mRNA was measured by quantifying chemokine and cytokine levels (i.e., IL-6 and MCP-1) from serum collected 4 hours after administration of the mRNA formulations using the MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead-Based Luminex Panel from EMD Millipore (Cat. No.: MCYTOMAG-70KM) (Figure 4F).

[0199] Overall, the in-house (J2 / dT) method, including poly(A) mRNA purification and dsRNA depletion, produced mRNA of equal or better quality than commercially available silica or HPLC purified mRNA in both in vitro mRNA characterization and in vivo activity and toxicity / immunogenicity assays.

[0200] Example 5 Ex vivo gene editing and mRNA characterization Crude in vitro transcribed (IVT) RNA material of PD1 megaTAL mRNA (SEQ ID NO:5) generated in-house was sent to a commercial vendor for capping and purification with either silica resin (commercial-silica) or HPLC (commercial-HPLC). The same crude IVT material was purified in-house using poly(A) mRNA isolation (oligo dT purification) and dsRNA depletion (J2 purification). The mRNA produced using the three methods was compared in three separate assays to assess the quality of the mRNA (Figures 5A-5C). The mRNA was also evaluated in T cells to compare the differences in efficacy (Figures 5D and 5E). PBMCs from three donors were stimulated with αCD3 and αCD28 antibodies. After 72 hours at 37°C, T cells were electroporated with mRNA using an Amaxa 4D-Nucleofector at a dose of 50 μg / mL. Each mRNA was electroporated in triplicate for each of the three donors. After electroporation, cells were placed at 30°C to recover overnight and then transferred to 37°C the next day. 96 hours after electroporation, cells were split into two plates. One plate was stimulated with PMA / ionomycin for 24 hours and then analyzed by FACS to observe PD-1 knockdown in PD1 megaTAL mRNA-treated cells (Figure 5E). The remaining plate was processed for INDEL analysis via NGS (Figure 5D).

[0201] Example 6 Comparison of dsRNA levels between different purification methods dsRNA levels were compared between mRNAs produced by different purification methods (Figure 6A). Multiple mRNA constructs were produced by commercial suppliers using their platform silica or HPLC purification processes (commercial silica or commercial HPLC). Additionally, a portion of each silica-purified mRNA was further purified using bluebird's J2 / dT process (commercial-J2). Two batches of in-house produced mRNA using the J2 / dT process were included in the comparison. Analysis showed that the additional J2 / dT purification surprisingly reduced the dsRNA levels of the silica-purified material. Furthermore, the in-house produced material purified by J2 / dT showed the lowest dsRNA levels.

[0202] In addition to dsRNA analysis by dot blot, in vitro cytotoxicity was assessed for a select group of mRNA materials by impedance-based assay using BJ fibroblasts. Cytotoxicity results, expressed as the slope of the cell proliferation index, showed a strong correlation with the dsRNA levels of the mRNA material (Figure 6B).

[0203] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. An RNA purification process comprising: (a) contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody that binds to dsRNA or an antigen-binding fragment thereof to form a dsRNA:antibody complex; (b) removing the dsRNA:antibody complex from the sample; (c) purifying the single-stranded RNA.

2. A process for producing therapeutic RNA, comprising: (a) contacting an RNA sample containing single-stranded RNA and double-stranded RNA (dsRNA) with an antibody that binds to dsRNA or an antigen-binding fragment thereof to form a dsRNA:antibody complex; (b) removing the dsRNA:antibody complex from the sample; (c) purifying the single-stranded RNA, thereby producing therapeutic RNA.

3. A process for increasing the efficiency of editing a nuclease, comprising: (a) contacting an RNA sample containing single-stranded RNA encoding a nuclease and double-stranded RNA (dsRNA) with an antibody that binds to dsRNA or an antigen-binding fragment thereof to form a dsRNA:antibody complex; (b) removing the dsRNA:antibody complex from the sample; (c) purifying the single-stranded RNA, wherein the rate of editing of the nuclease is increased as compared to the rate of editing of a nuclease encoded by RNA that does not contact an antibody that binds to dsRNA.

4. A process for reducing the immunogenicity and / or toxicity of RNA administered to a cell or subject, comprising: (a) contacting an RNA sample comprising single-stranded RNA and double-stranded RNA (dsRNA) with an antibody that binds to dsRNA or an antigen-binding fragment thereof to form a dsRNA:antibody complex; (b) removing the dsRNA:antibody complex from the sample; (c) purifying the single-stranded RNA, comprising a process wherein the immunogenicity and / or toxicity of the RNA when administered to a cell or subject is lower than the immunogenicity and / or toxicity of the RNA administered to a cell or subject when the RNA has not been contacted with an antibody that binds to dsRNA. A process according to any one of claims 1 to 4, wherein the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA.

5. A process according to any one of claims 1 to 4, wherein the single-stranded RNA is polyadenylated and / or the process comprises a polyadenylation step prior to contacting the sample with an antibody that binds to dsRNA or an antigen-binding fragment thereof.

6. A process according to any one of claims 1 to 4, wherein the process comprises contacting the polyadenylated RNA sample with a first oligonucleotide dT (oligo dT) probe that binds to polyadenylated RNA and removing unbound RNA from the sample prior to contacting the sample with an antibody that binds to dsRNA or an antigen-binding fragment thereof and / or the process further comprises contacting the polyadenylated RNA with a second oligonucleotide dT probe after contacting with the antibody that binds to dsRNA or an antigen-binding fragment thereof.

7. A process according to claim 6, wherein the process comprises contacting the polyadenylated RNA sample with a first oligonucleotide dT (oligo dT) probe that binds to polyadenylated RNA and removing unbound RNA from the sample prior to contacting the sample with an antibody that binds to dsRNA or an antigen-binding fragment thereof and / or the process further comprises contacting the polyadenylated RNA with a second oligonucleotide dT probe after contacting with the antibody that binds to dsRNA or an antigen-binding fragment thereof.

8. The process according to any one of claims 1 to 4, wherein the RNA sample is newly obtained through chemical synthesis or obtained from an in vitro transcription reaction, and / or the cytotoxicity of the purified RNA measured by impedance when administered to cells is lower than the cytotoxicity of the RNA administered to cells when the RNA has not contacted an antibody that binds to dsRNA and / or a second oligo dT.

9. The process according to claim 7, wherein the first and / or second oligonucleotide dT probe binds to a surface.

10. The process according to any one of claims 1 to 4, wherein the RNA in the sample is capped and / or the process includes capping the RNA in the sample, and / or the RNA is obtained from an in vitro transcription reaction and is co-transcriptionally capped.

11. The process according to claim 10, wherein the cap is cap 0 or cap 1, and / or the cap is an ARCA cap or a modified ARCA cap.

12. The process according to any one of claims 1 to 4, wherein the RNA in the sample is capped at its 5' end using a capping enzyme, guanosine triphosphate, and S-adenosyl-L-methionine.

13. The process according to claim 12, wherein the capping enzyme is vaccinia guanylyltransferase.

14. The process according to claim 10, wherein the capping includes S-adenosyl-L-methionine and / or the capping includes 2'-O-methyltransferase.

15. An antibody or antigen-binding fragment thereof that binds to the dsRNA is selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, camelid VHH, nanobody), and / or the antibody is selected from the group consisting of J2, J5, K1, K2, 1D3, CABT-B212, and 9D5. The process according to any one of claims 1 to 4.

16. The antibody or antigen-binding fragment thereof that binds to the dsRNA is a monoclonal antibody. The process according to any one of claims 1 to 4.

17. The antibody is J2. The process according to any one of claims 1 to 4.

18. The RNA contacts at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%, at least about 5.5 mol%, at least about 6 mol%, at least about 6.5 mol%, at least about 7 mol%, at least about 7.5 mol%, at least about 15 mol%, at least about 30 mol%, or at least about 60 mol% of the antibody compared to the total moles of RNA in the sample. The process according to any one of claims 1 to 4.

19. The sample contacts about 1.5 mol% to about 60 mol% of the antibody compared to the total moles of RNA in the sample. The process according to any one of claims 1 to 4.

20. The dsRNA:antibody complex is separated from the single-stranded RNA by antibody-based affinity chromatography, and / or the process includes a plasmid digestion step prior to the IVT step, and / or the process further includes a step of treating the sample with DNase to remove residual plasmid DNA template, and / or the process further includes one or more ultrafiltration / diafiltration steps, and / or the process further includes a final sterile filtration step, the process according to any one of claims 1 to 4.

21. The antibody-based affinity chromatography includes a 1 ml column, a 5 ml column, or a 10 ml column, the process according to claim 20.

22. The step of DNase treatment occurs after the IVT step and / or after the capping step, the process according to claim 20.

23. The ultrafiltration / diafiltration step is after the plasmid digestion step, the in vitro transcription step, the cap reaction step, or the affinity chromatography step, the process according to claim 20.

24. The nuclease is an endonuclease or an exonuclease, and / or the nuclease is a homing endonuclease, megaTAL, a CRISPR-related nuclease, a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN), the process according to claim 3.

25. The aspartate aminotransferase enzyme (AST) level in a subject administered with the purified RNA is lower than the AST level in a subject administered with purified RNA that has not been contacted with an antibody that binds to dsRNA and / or a second oligo dT, and / or the IL-6 level in a subject administered with the purified RNA is lower than the IL-6 level in a subject administered with purified RNA that has not been contacted with an antibody that binds to dsRNA and / or a second oligo dT, and / or the MCP-1 level in a subject administered with the purified RNA is lower than the MCP-1 level in a subject administered with purified RNA that has not been contacted with an antibody that binds to dsRNA and / or a second oligo dT, the process according to any one of claims 1 to 4.

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