SINGLE STRANDED RNA PURIFICATION METHODS
The present disclosure relates to improved therapeutic RNA compositions. More particularly, the disclosure relates to improved methods for purifying therapeutic RNA and related therapeutic RNA preparations.
1 . An RNA purification process comprising:
(a) contacting an RNA sample comprising single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen binding fragment thereof that binds dsRNA, thus forming dsRNA:antibody complexes;
(b) removing the dsRNA:antibody complexes from the sample; and
(c) purifying the single-stranded RNA.
2 . A process for producing therapeutic RNA comprising:
(a) contacting an RNA sample comprising single-stranded RNA and double-stranded RNA (dsRNA) with an antibody or antigen binding fragment thereof that binds dsRNA, thus forming dsRNA:antibody complexes;
(b) removing the dsRNA:antibody complexes from the sample; and
(c) purifying the single-stranded RNA;
thereby producing therapeutic RNA.
3 . A process for increasing nuclease editing efficiency comprising:
(a) contacting an RNA sample comprising single-stranded RNA and double-stranded RNA (dsRNA) encoding a nuclease with an antibody or antigen binding fragment thereof that binds dsRNA, thus forming dsRNA:antibody complexes;
(b) removing the dsRNA:antibody complexes from the sample; and
(c) purifying the single-stranded RNA;
wherein the editing rate of the nuclease is increased compared to the editing rate of a nuclease encoded by an RNA that is not contacted with an antibody that binds dsRNA.
4 . A process for decreasing 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 or antigen binding fragment thereof that binds dsRNA, thus forming dsRNA:antibody complexes;
(b) removing the dsRNA:antibody complexes from the sample; and
(c) purifying the single-stranded RNA;
wherein the immunogenicity and/or toxicity of the RNA when administered to a cell or subject is less than the immunogenicity and/or toxicity of an RNA administered to a cell or subject when the RNA has not been contacted with an antibody that binds dsRNA.
5 . The process of claim 4 , wherein the subject is human.
6 . The process of any one of claims 1-5 , wherein the single-stranded RNA is single-stranded circular RNA, single-stranded mRNA, or single-stranded non-coding RNA.
7 . The process of any one of claims 1-6 , wherein the single-stranded RNA is polyadenylated and/or the process comprises a polyadenylation step prior to contacting the sample with an antibody or antigen binding fragment thereof that binds dsRNA.
8 . The process of claim 7 , wherein the process comprises contacting the polyadenylated RNA sample with a first oligonucleotide dT (oligo dT) probe that binds polyadenylated RNA and removing unbound RNA from the sample prior to contacting the sample with an antibody or antigen binding fragment thereof that binds dsRNA.
9 . The process of claim 7 or claim 8 , wherein the process further comprises contacting the polyadenylated RNA with a second oligonucleotide dT probe after contacting with the antibody or antigen binding fragment thereof that binds dsRNA.
10 . The process of any one of claims 1-9 , wherein the RNA sample is obtained de novo through chemical synthesis.
11 . The process of any one of claims 1-9 , wherein the RNA sample is obtained from an in vitro transcription reaction.
12 . The process of any one of claims 1-11 , wherein cytotoxicity, as measured by impedance, of the purified RNA when administered to a cell is less than the cytotoxicity of RNA administered to a cell, when the RNA has not been contacted with an antibody that binds dsRNA and/or a second oligo dT.
13 . The process of any one of claims 8-12 , wherein the first and/or second oligonucleotide dT probe is bound to a surface.
14 . The process of claim 13 , wherein the first and/or second oligonucleotide dT probe is covalently linked to the surface.
15 . The process of any one of claims 1-14 , wherein the RNA in the sample is capped and/or the process comprises capping the RNA in the sample.
16 . The process of any one of claims 1-15 , wherein the RNA is obtained from an in vitro transcription reaction and is co-transcriptionally capped.
17 . The process of claim 15 or claim 16 , wherein the cap is a cap0 or cap1.
18 . The process of claim 15 or claim 16 , wherein the cap is an ARCA cap or modified ARCA cap.
19 . The process of any one of claims 1-18 , wherein the RNA in the sample is capped at its 5′ end using a capping enzyme, guanosine triphosphate, and S-adenosyl-L-methionine.
20 . The process of claim 19 , wherein the capping enzyme is Vaccinia guanylyltransferase.
21 . The process of claim 19 or claim 20 , wherein the capping comprises guanosine triphosphate.
22 . The process of any one of claim 15-21 , wherein the capping comprises S-adenosyl-L-methionine.
23 . The process of any one of claims 15-22 , wherein the capping comprises a 2′-O-Methyltransferase.
24 . The process of any one of claims 1-23 , wherein the antibody or antigen binding fragment thereof that binds dsRNA is selected from the group consisting of: a Camel Ig, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab′ fragment, a F(ab′)2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH, Nanobody).
25 . The process of any one of claims 1-24 , wherein the antibody or antigen binding fragment thereof that binds dsRNA is a monoclonal antibody.
26 . The process of any one of claims 1-25 , wherein the antibody is selected from the group consisting of: J2, J5, K1, K2, 1D3, CABT-B212, and 9D5.
27 . The process of any one of claims 1-26 , wherein the antibody is J2.
28 . The process of any one of claims 1-27 , wherein the RNA is contacted with at least about 1.5 mol %, at about least 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 % antibody, compared to total moles of RNA within the sample.
29 . The process of any one of claims 1-27 , wherein the RNA 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 % antibody, compared to total moles of RNA within the sample.
30 . The process of any one of claims 1-27 , wherein the sample is contacted with at least about 7.5 mol % antibody, compared to total moles of RNA within the sample.
31 . The process of any one of claims 1-27 , wherein 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 % antibody, compared to total moles of RNA within the sample.
32 . The process of any one of claims 1-27 , wherein the sample is contacted with about 7.5 mol % antibody, compared to total moles of RNA within the sample.
33 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
34 . The process of any one of claim 1-27 , wherein the sample is contacted with about 2 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
35 . The process of any one of claims 1-27 , wherein the sample is contacted with about 2.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
36 . The process of any one of claims 1-27 , wherein the sample is contacted with about 3 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
37 . The process of any one of claims 1-27 , wherein the sample is contacted with about 3.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
38 . The process of any one of claims 1-27 , wherein the sample is contacted with about 4 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
39 . The process of any one of claims 1-27 , wherein the sample is contacted with about 4.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
40 . The process of any one of claims 1-27 , wherein the sample is contacted with about 5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
41 . The process of any one of claims 1-27 , wherein the sample is contacted with about 5.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
42 . The process of any one of claims 1-27 , wherein the sample is contacted with about 6 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
43 . The process of any one of claims 1-27 , wherein the sample is contacted with about 6.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
44 . The process of any one of claims 1-27 , wherein the sample is contacted with about 7 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
45 . The process of any one of claims 1-27 , wherein the sample is contacted with about 7.5 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
46 . The process of any one of claims 1-27 , wherein the sample is contacted with about 15 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
47 . The process of any one of claims 1-27 , wherein the sample is contacted with about 30 mol % to about 60 mol % antibody, compared to total moles of RNA within the sample.
48 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 30 mol % antibody, compared to total moles of RNA within the sample.
49 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 15 mol % antibody, compared to total moles of RNA within the sample.
50 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 7.5 mol % antibody, compared to total moles of RNA within the sample.
51 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 7 mol % antibody, compared to total moles of RNA within the sample.
52 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 6.5 mol % antibody, compared to total moles of RNA within the sample.
53 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 6 mol % antibody, compared to total moles of RNA within the sample.
54 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 5.5 mol % antibody, compared to total moles of RNA within the sample.
55 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 5 mol % antibody, compared to total moles of RNA within the sample.
56 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 4.5 mol % antibody, compared to total moles of RNA within the sample.
57 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 4 mol % antibody, compared to total moles of RNA within the sample.
58 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 3.5 mol % antibody, compared to total moles of RNA within the sample.
59 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 3 mol % antibody, compared to total moles of RNA within the sample.
60 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 2.5 mol % antibody, compared to total moles of RNA within the sample.
61 . The process of any one of claims 1-27 , wherein the sample is contacted with about 1.5 mol % to about 2 mol % antibody, compared to total moles of RNA within the sample.
62 . The process of any one of claims 1 to 61 , wherein the dsRNA:antibody complex is separated from the single-stranded RNA by antibody-based affinity chromatography.
63 . The process of claim 62 , wherein the antibody-based affinity chromatography comprises a 1 ml column.
64 . The process of claim 62 , wherein the antibody-based affinity chromatography comprises a 5 ml column.
65 . The process of claim 62 , wherein the antibody-based affinity chromatography comprises a 10 ml column.
66 . The process of any one of the preceding claims , wherein the process comprises a plasmid digestion step prior to an IVT step.
67 . The process of any one of the preceding claims , wherein the process further comprises a step of treating the sample with a DNase to remove residual plasmid DNA template.
68 . The process of claim 67 , wherein the DNase treatment step occurs after an IVT step and/or after a capping step.
69 . The process of any one of the preceding claims , further comprising one or more ultrafiltration/diafiltration steps.
70 . The process of claim 69 , wherein the UF/DF step is after a plasmid digestion step, an in vitro transcription step, a cap reaction step, or an affinity chromatography step (e.g., dT or J2).
71 . The process of any one the preceding claims , further comprising a final sterile filtration step.
72 . The process of claim 71 , wherein the final sterile filtration step comprises filtration through a 0.22 μm filter.
73 . The process of any one of claims 3 and 6-72 , wherein the nuclease is an endonuclease or exonuclease.
74 . The process of any one of claims 3 and 6-73 , wherein the nuclease is a homing endonuclease, megaTAL, CRISPR-associated nuclease, zinc finger nuclease, transcription activator-like effector nuclease (TALEN).
75 . The process of claim 74 , wherein the CRISPR-associated nuclease is Cas9 or a variant thereof.
76 . The process of any one of claims 1-75 , wherein Aspartate Aminotransferase enzyme (AST) levels in a subject administered the purified RNA are less than AST levels in a subject administered purified RNA not contacted with an antibody that binds dsRNA and/or a second oligo dT.
77 . The process of any one of claims 1-76 , wherein IL-6 levels in a subject administered the purified RNA are less than IL-6 levels in a subject administered purified RNA not contacted with an antibody that binds dsRNA and/or a second oligo dT.
78 . The process of any one of claims 1-77 , wherein MCP-1 levels in a subject administered the purified RNA are less than MCP-1 levels in a subject administered purified RNA not contacted with an antibody that binds dsRNA and/or a second oligo dT.