JP2020534021A - 遺伝子治療DNAベクターVTvaf17と生産方法;大腸菌株SCS110−AFと生産方法;遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17と生産方法 - Google Patents
遺伝子治療DNAベクターVTvaf17と生産方法;大腸菌株SCS110−AFと生産方法;遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17と生産方法 Download PDFInfo
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Abstract
【選択図】図1
Description
I)遺伝子治療DNAベクターに抗生物質耐性遺伝子が存在しないためにヒトおよび動物の遺伝子治療に安全に使用できる可能性;
II)標的細胞への効率的遺伝子送達を保証する長さ;
III)標的遺伝子の効率的発現を保証し、しかもウイルスゲノムのヌクレオチド配列が呈するものでない調節要素(調節エレメント、制限エレメント/regulatory element)の存在;
IV)工業規模での生産可能性および構築可能性。
固有エンハンサーを伴うヒト伸張因子遺伝子EF1Aのプロモーター、ポリリンカー、ヒト増殖因子のポリアデニル化配列及び転写ターミネーター、トランスポゾンTn10の調節要素RNA−OUT、ならびにプラスミド生産を増加させるための一塩基置換を伴う複製起点を含有する遺伝子治療DNAベクターVTvaf17の生産。
(а)複製起点を、オリゴヌクレオチドOri−F、Ori−R、Ori−M1、およびOri−M2(配列表(1)〜(4))を用い、点突然変異を有する市販プラスミドpBR322の領域のPCR増幅によって生産し;
(b)プロモーター領域EF1аを、オリゴヌクレオチドEF1−FおよびEF1−R(配列表(5)および(6))を用い、ヒトゲノムDNAの部位のPCR増幅によって生産し;
(c)転写ターミネーターhGH−TAを、オリゴヌクレオチドhGH−FおよびhGH−R(配列表(7)および(8))を用い、ヒトゲノムDNAの部位のPCR増幅によって生産し;
(d)トランスポゾンTn10の調節部位RNA−OUTを、オリゴヌクレオチドRO−F、RO−R、RO−1、RO−2、およびRO−3(配列表(9)〜(13))から合成し;
(e)カナマイシン耐性遺伝子を、オリゴヌクレオチドKan−FおよびKan−R(配列表(14)および(15))を用い、市販プラスミドpET−28の部位のPCR増幅によって生産し;
(f)ポリリンカーを、2つの合成オリゴヌクレオチドMCS1およびMCS2(配列表(15)および(16))をアニーリングすることによって生産した。
DNAベクターVTvaf17の効率を証明するために、標的遺伝子、例えば、緑色蛍光タンパク質(GFP)遺伝子を、ポリリンカーにクローニングした。
DNAベクターVTvaf17の効率を証明するために、標的遺伝子、例えば、ヒトエラスチンコード遺伝子をポリリンカーにクローニングした。
遺伝子治療DNAベクターVTvaf17およびそれに基づく遺伝子治療ベクターを生産するための大腸菌株SCS110−AFの操作。
さらなる生産のための、遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17(番号B−12990、国際寄託当局番号NCIMB42801としてロシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオーガニズムに登録)の構築。
遺伝子治療DNAベクターVTvaf17の効率を証明するために、標的遺伝子、例えば、緑色蛍光タンパク質(GFP)コード遺伝子をポリリンカーにクローニングした。
Σタンパク質含量(μg)={[x]−σ}÷k*M
式中、[x]は各サンプルに関する4反復のOD620の平均値であり、σは平均偏差であり、kはBSAの検量線の傾斜係数であり、Mはサンプルの希釈倍率である。
OEn=[OE]÷Σタンパク質含量(mg)
式中、[ОЕ]は各サンプルに関する4反復の平均相対蛍光単位(RFU)である。
遺伝子治療DNAベクターVTvaf17の効率を証明するために、標的遺伝子、例えば、エラスチンコード遺伝子をポリリンカーにクローニングした。
1−遺伝子治療ベクターVTvaf17でのトランスフェクション後のELN遺伝子のcDNA;
2−エラスチン遺伝子コード領域を保持する遺伝子治療ベクターVTvaf17−ELNでのトランスフェクション後のELN遺伝子のcDNA;
3−遺伝子治療ベクターVTvaf17でのトランスフェクション後のB2M遺伝子のcDNA;
4−エラスチン遺伝子コード領域を保持する遺伝子治療ベクターVTvaf17−ELNでのトランスフェクション後のB2M遺伝子のcDNA。
遺伝子治療DNAベクターVTvaf17の効率を証明するために、標的遺伝子、例えば、エラスチンコード遺伝子をポリリンカーにクローニングした。
遺伝子治療DNAベクターVTvaf17の効率を証明するために、ウシ腎臓細胞MDBKにおいて標的遺伝子、例えば、緑色蛍光タンパク質(GFP)コード遺伝子をポリリンカーにクローニングした。
工業規模での遺伝子治療DNAベクターVTvaf17の生産可能性および構築可能性を証明するために、遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17(番号B−12990、国際寄託当局番号NCIMB42801としてロシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオーガニズムに登録)の大規模発酵を行った。
I)遺伝子治療DNAベクターに抗生物質耐性遺伝子が存在しないためにヒトおよび動物の遺伝子治療に安全に使用できる可能性;
II)標的細胞への効率的遺伝子送達を保証する長さ;
III)標的遺伝子の効率的発現を保証し、しかもウイルスゲノムのヌクレオチド配列が呈するものでない調節要素の存在;および
IV)工業規模での生産可能性および構築可能性
を合理的に兼ね備えている本発明の目的、具体的には、ヒトおよび動物細胞の遺伝子改変のための遺伝子治療DNAベクターの構築が達成され、このことは以下の実施例:項目I−実施例1;項目II−実施例1、6、7、8、9;項目III−実施例1、6、7、8、9;項目IV−実施例5、10により裏づけられる。
(1) オリゴヌクレオチドORI−F AGTCCATGGCTGCCTCGCGCGTTTCG
(2) オリゴヌクレオチドORI−R
AGCCTCACGGGAGTCAGGCAACTATG
(3) オリゴヌクレオチドOri−M1
CTACACTAGAAGAACAGTATTTG
(4) オリゴヌクレオチドOri−M2
CAAATACTGTTCTTCTAGTGTAG
(5) オリゴヌクレオチドEF1−F
CCTGACTCCCGTGAGGCTCCGGTGCC
(6) オリゴヌクレオチドEF1−R
TCGGATCCTGGCTTTTAGGGGTAGTTTTC
(7) オリゴヌクレオチドhGH−F
AGGATCCGAATTCCCTGTGACCCCTCCCCAG
(8) オリゴヌクレオチドhGH−R
CTCTTTACCAATTCTACGCGTAAGGACAGGGAAGGGAGCA
(9) オリゴヌクレオチドRO−F
CTTCCCTGTCCTTACGCGTAGAATTGGTAAAGAGAGTCGT
(10) オリゴヌクレオチドRO−R
CCGTAGAAAACTAGTTAATGATTTTTATCAAAATCATTAAG
(11) オリゴヌクレオチドRO−1
GAATTGGTAAAGAGAGTCGTGTAAAATATCGAGTTCGCACATCTTGTTG
(12) オリゴヌクレオチドRO−2
GATTTTTGGCGAAACCATTTGATCATATGACAAGATGTGTATCTACC
(13) オリゴヌクレオチドRO−3
ATGATTTTTATCAAAATCATTAAGTTAAGGTAGATACACATCTTGTC
(14) オリゴヌクレオチドKan−F
AAATCATTAACTAGTTTTCTACGGGGTCTGACGC
(15) オリゴヌクレオチドKan−R
CAGCCATGGACTAGTGGTGGCACTTTTCGGGGA
(16) オリゴヌクレオチドMCS1
GATCCGATATCGTCGACAAGCTTGGTACCG
(17) オリゴヌクレオチドMCS2
AATTCGGTACCAAGCTTGTCGACGATATCG
(18) オリゴヌクレオチドLHA−F 5’−
GCTGACGCTGCAGGTGATC
(19) オリゴヌクレオチドLHA−R 5’−
GACAAGATGTGTGTCTACCGCTTCAGGTTACCCGCCAG
(20) オリゴヌクレオチドIN−F 5’−
CTGGCGGGTAACCTGAAGCGGTAGACACACATCTTGTC
(21) オリゴヌクレオチドIN−1 5’−
ATTTTTGGCGAAACCATTCTATCATATGACAAGATGTGTGTC
(22) オリゴヌクレオチドIN−2 5’−
ATATGATAGAATGGTTTCGCCAAAAATCAATAATCAGACAAC
(23) オリゴヌクレオチドIN−R 5’−
CAAACTTTTTGATGTTCATCTTGTTGTCTGATTATTG
(24) オリゴヌクレオチドSACB−F 5’−
CAATAATCAGACAACAAGATGAACATCAAAAAGTTTG
(25) オリゴヌクレオチドSACB−R 5’−
CTTACGTGCCGATCATTATTTGTTAACTGTTAATTGTC
(26) オリゴヌクレオチドCATR−F 5’−
CAATTAACAGTTAACAAATAATGATCGGCACGTAAGAGG
(27) オリゴヌクレオチドCATR−R 5’−
CGAGACGAACAGAGGCGTAGTTACGCCCCGCCCTGCCAC
(28) オリゴヌクレオチドRHA−F 5’−
TGGCAGGGCGGGGCGTAACTACGCCTCTGTTCGTCTCGA
(29) オリゴヌクレオチドRHA−R 5’−
CTCAGCAGCAACTCACGTAC
(30) オリゴヌクレオチドMVGFP−F 5’−
GGATCCATGGTGAGCAAGGGCGAGGAGCT
(31) オリゴヌクレオチドMVGFP−R 5’−
GAATCCTCACAAATTTTGTAATCCAGAG
(32) オリゴヌクレオチドELN−F 5’−
GGATCCATGGCGGGTCTGACGG
(33) オリゴヌクレオチドELN−R 5’−
GAATCCTCATTTTCTCTTCCGGCCAC
(34) オリゴヌクレオチドEL1 F 5’−
GAGTCCTCCTGCTCCTGCTGTCCAT
(35) オリゴヌクレオチドEL1 R 5’−
AAAGGTAACTGCGGGGAAGGCG
VTvaf17 − ウイルスゲノムおよび抗生物質耐性マーカーの配列を含まない遺伝子治療ベクター(vector therapeutic virus−antibiotic−free)
DNA − デオキシリボ核酸
cDNA − 相補的デオキシリボ核酸
RNA − リボ核酸
mRNA − メッセンジャーリボ核酸
bp − 塩基対
PCR − ポリメラーゼ連鎖反応
ml − ミリリットル、μl − マイクロリットル
l − リットル
μg − マイクログラム
mg − ミリグラム
g − グラム
μmol − マイクロモル
mM − ミリモル
min − 分
s − 秒
rpm − 毎分回転数
nm − ナノモル
cm − センチメートル
mW − ミリワット
RFU − 相対蛍光単位
PBS − リン酸緩衝生理食塩水
Claims (6)
- 配列番号1のヌクレオチド配列を含有する動物およびヒト細胞の遺伝子改変のための3165bpの遺伝子治療DNAベクターVTvaf17。
- 3165bpの遺伝子治療DNAベクターVTvaf17を構築する方法であって、まず第一に、固有エンハンサーを伴うヒト伸張因子EF1Aの1188bpのプロモーター領域、制限エンドヌクレアーゼBamHI、EcoRV、SalI、HindIII、KpnI、EcoRIに対する部位を備えた35bpのポリリンカー、ヒト増殖因子の466bpのポリアデニル化配列及び転写ターミネーター、抗生物質を用いない正の選択を可能とするトランスポゾンTn10の136bpの調節要素RNA−OUT、ほとんどの大腸菌株の細胞においてベクター生産を増加させる一塩基置換を伴う自律的複製のための1299bpの複製起点、1010bpのカナマイシン耐性遺伝子を含有する4182bpのベクターを構築すること、次に、それがSpeI制限部位で切断され、残りの断片がそれ自体に連結されることを含む、方法。
- 抗生物質を用いない正の選択を可能とする遺伝子治療DNAベクターVTvaf17またはそれに基づく遺伝子治療DNAベクターを生産するための大腸菌株SCS110−AF。
- 抗生物質を用いない正の選択を可能とするトランスポゾンTn10の調節要素RNA−INを含有する64bpの直鎖DNA断片、産物がスクロース含有培地内での選択を保証する1422bpのレバンスクラーゼ遺伝子sacB、相同組換えが生じるクローン株のピッキングに必要とされる763bpのクロラムフェニコール耐性遺伝子catR、および遺伝子recAの領域での遺伝子の不活化を伴った相同組み換えを保証する2つの相同配列329bpと233bpを構築すること、次いで、大腸菌細胞をエレクトロポレーションにより形質転換させ、10μg/mlのクロラムフェニコールを含有する培地で生残しているクローンがピッキングされることを含む、遺伝子治療DNAベクターVTvaf17またはそれに基づく遺伝子治療DNAベクターを生産するための大腸菌株SCS110−AFを得る方法。
- 抗生物質を用いない選択を可能とするさらなる生産のための、遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17(番号В−12990、国際寄託当局No.NCIMB42801としてロシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオーガニズムに登録)。
- 遺伝子治療DNAベクターVTvaf17を保持する大腸菌株SCS110−AF/VTvaf17(番号В−12990、国際寄託当局No.NCIMB42801としてロシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオーガニズムに登録)を得る方法であって、大腸菌株SCS110−AFのエレクトロコンピテントセルを作出すること、およびこれらの細胞に遺伝子治療DNAベクターVTvaf17でエレクトロポレーションを行うこと、および、その後、これらの細胞がイーストレル、ペプトン、6%スクロース、および10μg/mlのクロラムフェニコールを含有する選択培地を含む寒天プレート(ペトリ皿)に注がれることを含む方法。
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RU2017130215A RU2678756C1 (ru) | 2017-08-25 | 2017-08-25 | Генотерапевтический ДНК-вектор VTvaf17, способ его получения, штамм Escherichia coli SCS110-AF, способ его получения, штамм Escherichia coli SCS110-AF/VTvaf17, несущий генотерапевтический ДНК-вектор VTvaf17, способ его получения |
RU2017130215 | 2017-08-25 | ||
PCT/RU2018/000191 WO2019039962A1 (en) | 2017-08-25 | 2018-03-26 | VTVAF17 GENE THERAPY DNA VECTOR, METHOD OF PRODUCTION; ESCHERICHIA COLI SCS110-AF STRAIN, PROCESS FOR PRODUCTION; VECTOR VTVAF17 GENE THERAPY DNA CARRYING THE ESCHERICHIA COLI SCS110-AF / VTVAF17 STRAIN, PRODUCTION PROCESS |
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CN113994005A (zh) * | 2018-12-27 | 2022-01-28 | 细胞基因治疗有限公司 | 基于基因疗法dna载体vtvaf17的基因疗法dna载体 |
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RU2705252C1 (ru) * | 2018-06-08 | 2019-11-06 | Селл энд Джин Терапи Лтд | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген CFTR, или NOS1, или AQ1, или AQ3, или AQ5, для лечения заболеваний, связанных с необходимостью повышения уровня экспрессии этих целевых генов, способ его получения и использования, штамм Escherichia coli SCS110-AF/VTvaf17-CFTR, или Escherichia coli SCS110-AF/VTvaf17-NOS1, или Escherichia coli SCS110-AF/VTvaf17-AQ1, или Escherichia coli SCS110-AF/VTvaf17-AQ3, или Escherichia coli SCS110-AF/VTvaf17-AQ5, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2707537C1 (ru) * | 2018-09-04 | 2019-11-27 | Общество с ограниченной ответственностью "Аллель Центр Инновационных Биотехнологий" | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген, выбранный из группы генов COL1A1, COL1A2, BMP2, BMP7, для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli SCS110-AF/VTvaf17- COL1A1 или Escherichia coli SCS110-AF/VTvaf17- COL1A2 или Escherichia coli SCS110-AF/VTvaf17- BMP2 или Escherichia coli SCS110-AF/VTvaf17- BMP7, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2712838C1 (ru) * | 2018-09-04 | 2020-01-31 | Генетик Диагностик Энд Терапи 21 Лтд | Генотерапевтический ДНК-вектор GDTT1.8NAS12, способ его получения, штамм Escherichia coli JM110-NAS, способ его получения, штамм Escherichia coli JM110-NAS/GDTT1.8NAS12, несущий генотерапевтический ДНК-вектор GDTT1.8NAS12, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2705256C1 (ru) * | 2018-09-05 | 2019-11-06 | Общество С Ограниченной Ответственностью "Прорывные Инновационные Технологии" | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора VTvaf17, несущий целевой ген, выбранный из группы генов SKI, TGFB3, TIMP2, FMOD для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli SCS110-AF/VTvaf17-SKI или Escherichia coli SCS110-AF/VTvaf17-TGFB3 или Escherichia coli SCS110-AF/VTvaf17-TIMP2 или Escherichia coli SCS110-AF/VTvaf17-FMOD, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2741570C1 (ru) * | 2019-12-26 | 2021-01-27 | Генетик Диагностикс Энд Терапи 21 Лтд | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора GDTT1.8NAS12, несущий целевой ген, выбранный из группы генов IFNB1, IFNA14, IFNA2, IL12A, IL12B для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli JM110-NAS/GDTT1.8NAS12-IFNB1, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IFNA14, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IFNA2, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IL12A, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IL12B, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2733429C1 (ru) * | 2019-12-30 | 2020-10-01 | Генетик Диагностикс Энд Терапи 21 Лтд | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора GDTT1.8NAS12, несущий целевой ген HFE для повышения уровня экспрессии этого целевого гена, способ его получения и применения, штамм Escherichia coli JM110-NAS/GDTT1.8NAS12-HFE несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
RU2734726C1 (ru) * | 2020-01-15 | 2020-10-22 | Генетик Диагностикс Энд Терапи 21 Лтд | Генотерапевтический ДНК-вектор на основе генотерапевтического ДНК-вектора GDTT1.8NAS12, несущий целевой ген, выбранный из группы генов DDC, IL10, IL13, IFNB1, TNFRSF4, TNFSF10, BCL2, HGF, IL2 для повышения уровня экспрессии этих целевых генов, способ его получения и применения, штамм Escherichia coli JM110-NAS/GDTT1.8NAS12-DDC, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IL10, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IL13, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IFNB1, или Escherichia coli JM110-NAS/GDTT1.8NAS12-TNFRSF4, или Escherichia coli JM110-NAS/GDTT1.8NAS12-TNFSF10, или Escherichia coli JM110-NAS/GDTT1.8NAS12-BCL2, или Escherichia coli JM110-NAS/GDTT1.8NAS12-HGF, или Escherichia coli JM110-NAS/GDTT1.8NAS12-IL2, несущий генотерапевтический ДНК-вектор, способ его получения, способ производства в промышленных масштабах генотерапевтического ДНК-вектора |
CN116162638A (zh) * | 2023-02-22 | 2023-05-26 | 中国科学院天津工业生物技术研究所 | 细菌中水平转移基因激活介导的基因串联重复的诱导方法 |
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GB201307075D0 (en) | 2013-04-19 | 2013-05-29 | Mayrhofer Peter | Plasmid for minicircle production |
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