JP6586075B2 - メッセンジャーrnaの精製方法 - Google Patents
メッセンジャーrnaの精製方法 Download PDFInfo
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- JP6586075B2 JP6586075B2 JP2016502790A JP2016502790A JP6586075B2 JP 6586075 B2 JP6586075 B2 JP 6586075B2 JP 2016502790 A JP2016502790 A JP 2016502790A JP 2016502790 A JP2016502790 A JP 2016502790A JP 6586075 B2 JP6586075 B2 JP 6586075B2
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- mrna
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- vitro
- rna
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- 150000002605 large molecules Chemical class 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 1
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 1
- MHCFAGZWMAWTNR-UHFFFAOYSA-M lithium perchlorate Chemical compound [Li+].[O-]Cl(=O)(=O)=O MHCFAGZWMAWTNR-UHFFFAOYSA-M 0.000 description 1
- 229910001486 lithium perchlorate Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- MJGFBOZCAJSGQW-UHFFFAOYSA-N mercury sodium Chemical compound [Na].[Hg] MJGFBOZCAJSGQW-UHFFFAOYSA-N 0.000 description 1
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- IZAGSTRIDUNNOY-UHFFFAOYSA-N methyl 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetate Chemical compound COC(=O)COC1=CNC(=O)NC1=O IZAGSTRIDUNNOY-UHFFFAOYSA-N 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- XJVXMWNLQRTRGH-UHFFFAOYSA-N n-(3-methylbut-3-enyl)-2-methylsulfanyl-7h-purin-6-amine Chemical compound CSC1=NC(NCCC(C)=C)=C2NC=NC2=N1 XJVXMWNLQRTRGH-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical group 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical class NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- RHFUOMFWUGWKKO-UHFFFAOYSA-N s2C Natural products S=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 RHFUOMFWUGWKKO-UHFFFAOYSA-N 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001023 sodium amalgam Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical class [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010036927 trypsin-like serine protease Proteins 0.000 description 1
- HDZZVAMISRMYHH-KCGFPETGSA-N tubercidin Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HDZZVAMISRMYHH-KCGFPETGSA-N 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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Description
本願は、2013年3月14日出願の米国仮特許出願第61/784,996号の利益を主張し、その文献の全体が参照により本明細書に援用される。
本発明は、例えば以下の項目を提供する。
(項目1)
メッセンジャーRNA(mRNA)の精製方法であって、
(a)生体外合成したmRNAを含む不純調製物を変性条件に付すことと、
(b)ステップ(a)による不純調製物から前記mRNAをタンジェント流濾過によって精製することとを含み、
ステップ(b)により精製した前記mRNAには、生体外合成で用いた未成熟中断RNA配列及び/または酵素試薬が実質的にない、
前記方法。
(項目2)
ステップ(a)が、前記不純調製物にタンパク質変性剤を添加することを含む、項目1に記載の方法。
[項目2a]
ステップ(a)が、前記タンパク質変性剤の添加された前記不純調製物を室温で約5分間インキュベートすることを含む、項目2に記載の方法。
(項目3)
前記タンパク質変性剤が、尿素、チオシアン酸グアニジン、KCl、ドデシル硫酸ナトリウム、サルコシル、及びその組合せからなる群より選択される、項目2または2aに記載の方法。
(項目4)
ステップ(a)が、前記不純調製物に尿素を添加して約1M以上の結果的尿素濃度を達成することを含む、項目1〜3のいずれか一項に記載の方法。
(項目5)
前記結果的尿素濃度が、約2M以上、3M以上、4M以上、5M以上、6M以上、7M以上、8M以上、9M以上、または10M以上である、項目4に記載の方法。
(項目6)
ステップ(a)が、前記不純調製物にチオシアン酸グアニジンを添加して約4M以上の結果的チオシアン酸グアニジン濃度を達成することを含む、項目1〜5のいずれか一項に記載の方法。
(項目7)
前記結果的チオシアン酸グアニジン濃度が、約4M以上、5M以上、6M以上、7M以上、8M以上、9M以上、または10M以上である、項目6に記載の方法。
(項目8)
ステップ(a)が、前記不純調製物にKClを添加して約1M以上の結果的KCl濃度を達成することを含む、項目1〜7のいずれか一項に記載の方法。
(項目9)
前記結果的KCl濃度が、約2M以上、3M以上、4M以上、または5M以上である、項目8に記載の方法。
(項目10)
前記タンジェント流濾過が、水性溶媒のみを用いて行われる、項目1〜9のいずれか一項に記載の方法。
(項目11)
前記タンジェント流濾過が、溶媒として水を用いて行われる、項目10に記載の方法。
(項目12)
ステップ(b)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を1%未満含有する、項目1〜11のいずれか一項に記載の方法。
(項目13)
ステップ(b)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を0.5%未満含有する、項目1〜12のいずれか一項に記載の方法。
(項目14)
ステップ(b)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を0.1%未満含有する、項目1〜13のいずれか一項に記載の方法。
(項目15)
ステップ(b)により精製した前記mRNAが、生体外合成で用いた未成熟中断したRNA配列及び/または酵素試薬を、アガロースゲル電気泳動法またはクロマトグラフィー法によって決定して検知できない程度に含有する、項目1〜14のいずれか一項に記載の方法。
(項目16)
前記未成熟中断したRNA配列が、15未満の塩基を含む、項目1〜15のいずれか一項に記載の方法。
(項目17)
前記未成熟中断したRNA配列が、約8〜12の塩基を含む、項目1〜16のいずれか一項に記載の方法。
(項目18)
生体外合成で用いた前記酵素試薬が、T7 RNAポリメラーゼ、デオキシリボヌクレアーゼI、ピロホスファターゼ、及び/またはリボヌクレアーゼ阻害薬を含む、項目1〜17のいずれか一項に記載の方法。
(項目19)
生体外合成で用いた前記酵素試薬が、T7 RNAポリメラーゼを含む、項目1〜18のいずれか一項に記載の方法。
(項目20)
前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加される前に行われる、項目1〜19のいずれか一項に記載の方法。
(項目21)
前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加された後に行われる、項目1〜19のいずれか一項に記載の方法。
(項目22)
前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加される前後の両方に行われる、項目1〜19のいずれか一項に記載の方法。
(項目23)
前記生体外合成したmRNAが、長さが約1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb、または5kbより大きい、項目1〜22のいずれか一項に記載の方法。
(項目24)
前記生体外合成したmRNAが、安定性を向上させるように1つまたは複数の修飾を含む、項目1〜23のいずれか一項に記載の方法。
(項目25)
前記1つまたは複数の修飾が、修飾されたヌクレオチド、修飾された糖リン酸塩主鎖、5’及び/または3’非翻訳領域からなる群より選択される、項目24に記載の方法。
(項目26)
前記生体外合成したmRNAが無修飾である、項目1〜23のいずれか一項に記載の方法。
(項目27)
ステップ(b)により精製した前記mRNAが、95%より大きい完全性を有する、項目1〜26のいずれか一項に記載の方法。
(項目28)
ステップ(b)により精製した前記mRNAが、98%より大きい完全性を有する、項目1〜27のいずれか一項に記載の方法。
(項目29)
ステップ(b)により精製した前記mRNAが、99%より大きい完全性を有する、項目1〜28のいずれか一項に記載の方法。
(項目30)
メッセンジャーRNA(mRNA)の製造方法であって、
mRNAを生体外で合成することと、
項目1〜29のいずれか一項に記載の方法を用いて前記生体外合成したmRNAを精製することと、
を含む前記方法。
(項目31)
項目1〜30のいずれか一項に記載の方法を用いて精製したメッセンジャーRNA(mRNA)。
本発明がより容易に理解されるように、まず所定の用語を下に定義する。次の用語及び他の用語に対する追加的定義も本明細書全体を通じて明記する。
本発明は、タンジェント流濾過に基づいて不純調製物(例えば、生体外合成反応混合物)からmRNAを精製する改善された方法をとりわけ提供する。いくつかの実施形態では、本発明による発明的方法は、(a)生体外合成したmRNAを含む不純調製物を変性条件に付すことと、(b)ステップ(a)から得た不純調製物からmRNAをタンジェント流濾過によって精製することとによるステップを含み、ステップ(b)により精製したmRNAには、生体外合成で用いた未成熟中断RNA配列及び/または酵素試薬が実質的にない。
mRNAの合成
変性条件及び変性剤
精製
従来式膜濾過
タンジェント流濾過
精製mRNAの特徴づけ
mRNAの合成
以下の実施例のそれぞれでは、mRNAの合成を完全なリボヌクレアーゼ非含有条件下で実施した。全てのチューブ、バイアル、ピペットチップ、ピペット、緩衝液等は、他に明示的に断りがなければ、ヌクレアーゼ非含有である必要があった。
5’キャップ及び3’尾部の付加
タンジェント流濾過による精製
実施例2.精製mRNAの分析
精製したmRNAにおける酵素の存在の試験
アガロースゲル電気泳動アッセイによるmRNA完全性の評価
生体外mRNA完全性アッセイ
バイオルミネセンス分析
実施例3.ホタルルシフェラーゼ(FFL)メッセンジャーRNA(mRNA)の生成と精製
コドン最適化ホタルルシフェラーゼ(FFL)mRNA(配列番号1)
X2AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAY2
5’及び3’UTR配列:
X2=
GGGAUCCUACC(配列番号2)
Y2=
UUUGAAUU(配列番号3)
FFL構築物:
1.TFF(尿素有り)によって精製したFFL IVT及びTFF(尿素無し)によるC/Tステップ
2.TFF(尿素有り)によって精製したFFL IVT及びTFF(尿素無し)によるC/Tステップ
3.スピンカラムによって精製したFFL IVT及びスピンカラムによるC/Tステップ
実施例4.因子IX(FIX)mRNAの生成と精製
ヒト因子IX(FIX)mRNA(配列番号4)
X1AUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCAUCUGCCUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUGAAAACGCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAGUUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAAGAAGCACGAGAAGUUUUUGAAAACACUGAAAGAACAACUGAAUUUUGGAAGCAGUAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUGGCGGCAGUUGCAAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAGAACUGUGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUUUGUAAAAAUAGUGCUGAUAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGACUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGAGUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUGUGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAAGCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAACCAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGCAUUCUGUGGAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUGUGUUGAAACUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAACAUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUGCAGCUAUUAAUAAGUACAACCAUGACAUUGCCCUUCUGGAACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGACAAGGAAUACACGAACAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCACAAAGGGAGAUCAGCUUUAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCGAGCCACAUGUCUUCGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCUGGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAUGUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAAGAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUCAACUGGAUUAAGGAAAAAACAAAGCUCACUUAAY1
5’及び3’UTR配列:
X1=
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(配列番号5)
Y1=
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(配列番号6)
実施例5.嚢胞性線維症膜コンダクタンス制御因子(CFTR)mRNAの生成及び生成
コドン最適化嚢胞性線維症膜コンダクタンス制御因子(CFTR)mRNA(配列番号7)
X1AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAY1
5’及び3’UTR配列:
X1=
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(配列番号5)
Y1=
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(配列番号6)
同等事項及び範囲
Claims (28)
- メッセンジャーRNA(mRNA)の精製方法であって、
(a)生体外合成したmRNA、未成熟中断RNA配列及びDNA鋳型を含む不純調製物を提供することと、
(b)前記mRNA及びDNA鋳型からの前記未成熟中断RNA配列の分離を促進する条件下で前記不純調製物をカオトロピック剤で処理することと、
(c)ステップ(b)により処理された不純調製物をタンジェント流濾過に付すことによってmRNAを精製することとを含み、
ステップ(c)により精製した前記精製mRNAには、未成熟中断RNAが実質的になく、前記精製mRNAが95%より大きい完全性を有する、
前記方法。 - ステップ(b)が、前記カオトロピック剤の添加された前記不純調製物を室温で約5分間インキュベートすることを含む、請求項1に記載の方法。
- 前記カオトロピック剤が、尿素、チオシアン酸グアニジン、ドデシル硫酸ナトリウム、及びその組合せからなる群より選択される、請求項1または2に記載の方法。
- ステップ(b)が、前記不純調製物に尿素を添加して約1M以上の結果的尿素濃度を達成することを含む、請求項1〜3のいずれか一項に記載の方法。
- 前記結果的尿素濃度が、約2M以上、3M以上、4M以上、5M以上、6M以上、7M以上、8M以上、9M以上、または10M以上である、請求項4に記載の方法。
- ステップ(b)が、前記不純調製物にチオシアン酸グアニジンを添加して約4M以上の結果的チオシアン酸グアニジン濃度を達成することを含む、請求項1〜3のいずれか一項に記載の方法。
- 前記結果的チオシアン酸グアニジン濃度が、約4M以上、5M以上、6M以上、7M以上、8M以上、9M以上、または10M以上である、請求項6に記載の方法。
- 前記タンジェント流濾過が、水性溶媒のみを用いて行われる、請求項1〜7のいずれか一項に記載の方法。
- 前記タンジェント流濾過が、溶媒として水を用いて行われる、請求項8に記載の方法。
- ステップ(c)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を1%未満含有する、請求項1〜9のいずれか一項に記載の方法。
- ステップ(c)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を0.5%未満含有する、請求項1〜10のいずれか一項に記載の方法。
- ステップ(c)により精製した前記mRNAが、未成熟中断したRNA配列及び/または生体外合成で用いた酵素試薬を0.1%未満含有する、請求項1〜11のいずれか一項に記載の方法。
- ステップ(c)により精製した前記mRNAが、生体外合成で用いた未成熟中断したRNA配列及び/または酵素試薬を、アガロースゲル電気泳動法またはクロマトグラフィー法によって決定して検知できない程度に含有する、請求項1〜12のいずれか一項に記載の方法。
- 前記未成熟中断したRNA配列が、15未満の塩基を含む、請求項1〜13のいずれか一項に記載の方法。
- 前記未成熟中断したRNA配列が、約8〜12の塩基を含む、請求項1〜14のいずれか一項に記載の方法。
- 生体外合成で用いた前記酵素試薬が、T7 RNAポリメラーゼ、デオキシリボヌクレアーゼI、ピロホスファターゼ、及び/またはリボヌクレアーゼ阻害薬を含む、請求項1〜15のいずれか一項に記載の方法。
- 生体外合成で用いた前記酵素試薬が、T7 RNAポリメラーゼを含む、請求項1〜16のいずれか一項に記載の方法。
- 前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加される前に行われる、請求項1〜17のいずれか一項に記載の方法。
- 前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加された後に行われる、請求項1〜17のいずれか一項に記載の方法。
- 前記タンジェント流濾過が、前記生体外合成したmRNAにキャップとポリA尾部が付加される前後の両方に行われる、請求項1〜17のいずれか一項に記載の方法。
- 前記生体外合成したmRNAが、長さが約1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb、または5kbより大きい、請求項1〜20のいずれか一項に記載の方法。
- 前記生体外合成したmRNAが、安定性を向上させるように1つまたは複数の修飾を含む、請求項1〜21のいずれか一項に記載の方法。
- 前記1つまたは複数の修飾が、修飾されたヌクレオチド、修飾された糖リン酸塩主鎖、5’及び/または3’非翻訳領域から選択される、請求項22に記載の方法。
- 前記生体外合成したmRNAが無修飾である、請求項1〜21のいずれか一項に記載の方法。
- ステップ(c)により精製した前記mRNAが、98%より大きい完全性を有する、請求項1〜24のいずれか一項に記載の方法。
- ステップ(c)により精製した前記mRNAが、99%より大きい完全性を有する、請求項1〜25のいずれか一項に記載の方法。
- メッセンジャーRNA(mRNA)の製造方法であって、
mRNAを生体外で合成することと、
請求項1〜26のいずれか一項に記載の方法を用いて前記生体外合成したmRNAを精製することと、
を含む前記方法。 - 請求項1〜27のいずれか一項に記載の方法を用いて精製したメッセンジャーRNA(mRNA)混合物であって、前記メッセンジャーRNAには、未成熟中断配列が実質的になく、前記メッセンジャーRNAが95%より大きい完全性を有する、メッセンジャーRNA(mRNA)混合物。
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UA117008C2 (uk) | 2013-03-14 | 2018-06-11 | Шир Хьюман Дженетік Терапіс, Інк. | IN VITRO ТРАНСКРИБОВАНА мРНК ТА КОМПОЗИЦІЯ, ЩО ЇЇ МІСТИТЬ, ДЛЯ ЗАСТОСУВАННЯ В ЛІКУВАННІ МУКОВІСЦИДОЗУ В ССАВЦЯ |
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EP3060303B1 (en) | 2013-10-22 | 2018-11-14 | Translate Bio, Inc. | Mrna therapy for argininosuccinate synthetase deficiency |
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EP4108769B1 (en) * | 2015-05-29 | 2023-08-30 | CureVac Manufacturing GmbH | A method for producing and purifying rna, comprising at least one step of tangential flow filtration |
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IL268857B2 (en) * | 2017-02-27 | 2024-09-01 | Translate Bio Inc | Methods for the purification of messenger RNA |
KR20210125536A (ko) * | 2019-02-11 | 2021-10-18 | 에트리스 게엠베하 | 접선 유동 여과에 의한 mRNA 정제 |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2018174942A (ja) * | 2013-03-14 | 2018-11-15 | トランスレイト バイオ, インコーポレイテッド | メッセンジャーrnaの精製方法 |
JP2022001056A (ja) * | 2013-03-14 | 2022-01-06 | トランスレイト バイオ, インコーポレイテッド | メッセンジャーrnaの精製方法 |
JP7166407B2 (ja) | 2013-03-14 | 2022-11-07 | トランスレイト バイオ, インコーポレイテッド | メッセンジャーrnaの精製方法 |
US11692189B2 (en) | 2013-03-14 | 2023-07-04 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11820977B2 (en) | 2013-03-14 | 2023-11-21 | Translate Bio, Inc. | Methods for purification of messenger RNA |
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