RU2020114699A - Эукариотическая клеточная линия - Google Patents
Эукариотическая клеточная линия Download PDFInfo
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Claims (26)
1. Клеточная линия, в которой тетрациклиновый репрессор (Tet-R) экспрессируется под контролем клеточного или частично клеточного промотора.
2. Клеточная линия по п. 1, в которой тетрациклиновый репрессор (Tet-R) экспрессируется по меньшей мере в 90% клеток клеточной линии.
3. Клеточная линия по п. 1 или 2, в которой экспрессия является стабильной во времени.
4. Клеточная линия по любому из пп. 1-3, в которой Tet-R репрессирует транскрипцию генов, содержащих в своих промоторах один или несколько тетрациклиновых операторов (TetO) в присутствии или в отсутствие тетрациклина или аналога тетрациклина.
5. Клеточная линия по любому из пп. 1-4, в которой ген Tet-R находится под контролем конститутивного промотора, предпочтительно конститутивный промотор выбран из группы, состоящей из промотора EF1 альфа, промотора PGK-1, промотора убиквитина В, промотора β-актина, промотора EGR1, промотора CASI и промотора CAG.
6. Клеточная линия по любому из пп. 1-5, в которой ген, кодирующий Tet-R, кодон-оптимизирован для экспрессии в эукариотических клетках, предпочтительно в клетках млекопитающего.
7. Клеточная линия по любому из пп. 1-6, которая дополнительно содержит:
(i) в своем геноме мутацию, делецию или вставку, которая уменьшает или предотвращает экспрессию функционального белка, выбранного из группы, состоящей из одного или нескольких стимуляторов гена IFN, предпочтительно cGas и STING, одной или нескольких сигнальных систем JAK/активатор транскрипции STAT, и одного или нескольких IFN-стимулированных генов (ISG), предпочтительно PKR, МХ1, OAS1, APOBEC3G, TRIM5, ZAP, ISG15, ADAR, IFITM1/2/3, BST2 и/или RSAD2, и/или
(ii) ген, кодирующий маркер селекции, предпочтительно ген устойчивости к G418, гигромицину В, пуромицину, бластицидину или зеоцину в генетической связи с геном, кодирующим Tet-R.
8. Клеточная линия по любому из пп. 1-7, содержащая стабильно интегрированный в ее геном ген, кодирующий тетрациклиновый репрессор (Tet-R).
9. Клеточная линия по любому из пп. 1-8, в которой клетки клеточной линии экспрессируют по меньшей мере один вирусный ген, который требуется для образования инфекционных вирусных частиц, предпочтительно при этом по меньшей мере один вирусный ген выбран из группы, состоящей из Е1, предпочтительно содержащей ген с нуклеотидной последовательностью SEQ ID NO: 1.
10. Клеточная линия по любому из пп. 1-9, где клетки клеточной линии представляют собой клетки человека, предпочтительно эмбриональные клетки почки человека, или отличающаяся тем, что клетки клеточной линии выбраны из группы, состоящей из клеток HEK293, клеток А549, клеток HeLa, клеток MRC5, клеток VERO, клеток DF-1 или клеток SK-OV-3.
11. Клеточная линия по любому из пп. 1-10, в которой промотор представляет собой промотор CAG.
12. Клеточная линия по любому из пп. 1-11, которая представляет собой клеточную линию, депонированную в ЕСАСС под номером доступа 17080901.
13. Клеточная линия по любому из пп. 1-12, дополнительно содержащая вирусный геном, способный к сборке в инфекционную вирусную частицу, и содержащая по меньшей мере один гетерологичный ген под контролем промотора, содержащего один или несколько тетрациклиновых операторов (TetO), предпочтительно при этом
(i) вирусный геном выбран из группы, состоящей из генома вируса герпеса, генома модифицированного вируса осповакцины Анкара и аденовирусного генома, предпочтительно вирусный геном представляет собой аденовирусный геном, и/или
(ii) гетерологичный ген является токсичным для клетки или нарушает цикл репликации вируса.
14. Клеточная линия по п. 13, отличающаяся тем, что гетерологичный ген выбран из группы, состоящей из аутоантигенов, вирусных генов, генов клеточного происхождения, синтетических цепочек, кодирующих комбинацию опухоль-ассоциированных неоантигенов, и цепочек, кодирующих неоэпитопы, предпочтительно при этом
(i) аутоантиген выбран из группы, состоящей из HER2/neu, СЕА, Нерсат, PSA, PSMA, теломеразы, gplOO, Melan-A/MART-1, Muc-1, NY-ESO-1, сурвивина, стромелизина 3, тирозиназы, MAGE3, CML6S, CML66, OY-TES-1, SSX-2, SART-1, SART-2, SART-3, NYC0-5S, NY-BR-62, hKLP2 и VEGF,
(ii) вирусный ген выбран из группы, состоящей из белка Е1-Е2 HCV, гликопротеина вируса бешенства и GP 160 HIV-1, и/или
iii) ген клеточного происхождения выбран из группы, состоящей из Tim-3, Her2 и Е2 F-1.
15. Применение клеточной линии по любому из пп. 1-14 для продуцирования инфекционных вирусных частиц.
16. Способ получения инфекционных вирусных частиц, включающий следующие стадии:
(i) выращивание клеток клеточной линии по п. 13 или 14 in vitro в присутствии или в отсутствие тетрациклина или аналога тетрациклина; или
(ii) инфицирование клеток клеточной линии по любому из пп. 1-12 вирусным вектором, предпочтительно аденовирусным вектором; и
(iii) извлечение вирусных частиц.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP17198339 | 2017-10-25 | ||
| EP17198339.8 | 2017-10-25 | ||
| PCT/EP2018/079341 WO2019081673A1 (en) | 2017-10-25 | 2018-10-25 | LINE OF EUKARYOTIC CELLS |
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| Publication Number | Publication Date |
|---|---|
| RU2020114699A3 RU2020114699A3 (ru) | 2021-11-25 |
| RU2020114699A true RU2020114699A (ru) | 2021-11-25 |
| RU2776531C2 RU2776531C2 (ru) | 2022-07-21 |
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| KR20200101322A (ko) | 2020-08-27 |
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| JP7274225B2 (ja) | 2023-05-16 |
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| IL274191A (en) | 2020-06-30 |
| WO2019081673A1 (en) | 2019-05-02 |
| JP2021500011A (ja) | 2021-01-07 |
| AU2018356538B2 (en) | 2023-09-28 |
| CN111386126A (zh) | 2020-07-07 |
| BR112020007772A2 (pt) | 2020-10-06 |
| KR102688005B1 (ko) | 2024-07-25 |
| CA3076026A1 (en) | 2019-05-02 |
| MX2020004305A (es) | 2020-11-11 |
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