RU2007131270A - Малые молекулы рнк, опосредующие интерференцию рнк - Google Patents

Малые молекулы рнк, опосредующие интерференцию рнк Download PDF

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RU2007131270A
RU2007131270A RU2007131270/13A RU2007131270A RU2007131270A RU 2007131270 A RU2007131270 A RU 2007131270A RU 2007131270/13 A RU2007131270/13 A RU 2007131270/13A RU 2007131270 A RU2007131270 A RU 2007131270A RU 2007131270 A RU2007131270 A RU 2007131270A
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rna molecule
gene
cell
nucleic acid
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Томас ТУШЛЬ (DE)
Томас ТУШЛЬ
Зайда ЭЛЬБАШИР (DE)
Зайда ЭЛЬБАШИР
Винфрид ЛЕНДЕККЕЛЬ (DE)
Винфрид ЛЕНДЕККЕЛЬ
Маттиас ВИЛЬМ (DE)
Маттиас ВИЛЬМ
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Макс-Планк-Гезелльшафт Цур Фердерунг Дер Виссеншафтен Е.Ф. (De)
Макс-Планк-Гезелльшафт Цур Фердерунг Дер Виссеншафтен Е.Ф.
Ойропэишес Лабораториум Фюр Молекулярбиологи (Ембл) (De)
Ойропэишес Лабораториум Фюр Молекулярбиологи (Ембл)
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Claims (47)

1. Выделенная двухцепочечная молекула РНК, где каждая цепь РНК имеет длину 19-25 нуклеотидов, причем указанная молекула РНК способна к мишень-специфическим модификациям нуклеиновых кислот.
2. Молекула РНК по п.1, где по меньшей мере одна цепь имеет 3'-выступ из 1-5 нуклеотидов.
3. Молекула РНК по п.1 или 2, способная к мишень-специфической интерференции РНК и/или метилированию ДНК.
4. Молекула РНК по любому из пп.1-3, где каждая цепь имеет длину 19-23, предпочтительно 20-22 нуклеотида.
5. Молекула РНК по любому из пп.2-4, где 3'-выступ состоит из 1-3 нуклеотидов.
6. Молекула РНК по любому из пп.2-5, где 3'-выступ стабилизирован против деградации.
7. Молекула РНК по любому из пп.1-6, которая содержит по меньшей мере один модифицированный аналог нуклеотида.
8. Молекула РНК по п.7, где модифицированный аналог нуклеотида выбран из рибонуклеотидов с модифицированным сахаром или скелетом молекулы.
9. Молекула РНК по п.7 или 8, где аналог нуклеотида является сахар-модифицированным рибонуклеотидом, где группа 2'-ОН заменена группой, выбранной из Н, OR, R, галогена, SH, SR1, NH2, NHR, NR2 или CN, где R обозначает С1-С6-алкил, алкенил или алкинил и галоген обозначает F, Cl, Br или I.
10. Молекула РНК по п.7 или 8, где аналог нуклеотида является скелет-модифицированным рибонуклеотидом, содержащим фосфотиоатную группу.
11. Молекула РНК по любому из пп.1-10, которая имеет последовательность, имеющую идентичность по меньшей мере 50% относительно заранее заданной молекулы мРНК-мишени.
12. Молекула РНК по п.11, где эта идентичность равна по меньшей мере 70%.
13. Способ получения двухцепочечной молекулы РНК любого из пп.1-12, предусматривающий стадии
(а) синтеза двух цепей РНК, каждая из которых имеет длину по меньшей мере 19-25 нуклеотидов, где указанные цепи РНК способны образовывать двухцепочечную молекулу РНК,
(b) объединения этих синтезированных цепей РНК в условиях, в которых образуется двухцепочечная молекула РНК, которая способна к мишень-специфическим модификациям нуклеиновых кислот.
14. Способ по п.13, где эти цепи РНК являются химически синтезированными.
15. Способ по п.13, где эти цепи РНК являются ферментативно синтезированными.
16. Способ опосредования мишень-специфических модификаций нуклеиновых кислот в клетке или организме, предусматривающий стадии
(а) контактирования указанной клетки или организма с двухцепочечной молекулой РНК любого из пп.1-12 в условиях, в которых могут происходить мишень-специфические модификации нуклеиновых кислот, и
(b) опосредования мишень-специфической модификации нуклеиновой кислоты, выполняемой этой двухцепочечной РНК в отношении нуклеиновой кислоты-мишени, имеющей часть последовательности, по существу соответствующую этой двухцепочечной РНК.
17. Способ по п.16, где модификация нуклеиновой кислоты является интерференцией РНК и/или метилированием ДНК.
18. Способ по пп.16 и 17, где указанное контактирование предусматривает введение указанной молекулы двухцепочечной РНК в клетку-мишень, в которой может происходить мишень-специфическая модификация нуклеиновой кислоты.
19. Способ по п.18, где это введение предусматривает опосредованную носителем доставку или инъекцию.
20. Применение способа по любому из пп.16-19 для определения функции гена в клетке или организме.
21. Применение способа по любому из пп.16-19 для модуляции функции гена в клетке или организме.
22. Применение по п.20 или 21, где этот ген ассоциирован с патологическим состоянием.
23. Применение по п.22, где этот ген является патогенассоциированным геном.
24. Применение по п.23, где этот ген является вирусным геном.
25. Применение по п.22, где этот ген является ассоциированным с опухолью геном.
26. Применение по п.22, где этот ген является ассоциированным с аутоиммунным заболеванием геном.
27. Фармацевтическая композиция, содержащая в качестве активного агента по меньшей мере одну двухцепочечную молекулу РНК по любому из пп.1-12 и фармацевтический носитель.
28. Композиция по п.27 для диагностических применений.
29. Композиция по п.27 для терапевтических применений.
30. Эукариотическая клетка или эукариотический организм (не человек), проявляющие фенотип со специфическим в отношении гена-мишени нокаутом, где указанные клетка или организм трансфицированы по меньшей мере одной двухцепочечной молекулой РНК, способной ингибировать экспрессию по меньшей мере одного эндогенного гена-мишени, или ДНК, кодирующей по меньшей мере одну молекулу двухцепочечной РНК, способную ингибировать экспрессию по меньшей мере одного эндогенного гена-мишени.
31. Клетка (или организм) по п.30, которая является клеткой млекопитающего.
32. Клетка (или организм) по п.31, которая является клеткой человека.
33. Клетка или организм по любому из пп.30-32, которые дополнительно трансфицированы по меньшей мере одной экзогенной нуклеиновой кислотой-мишенью, кодирующей белок-мишень, или вариант, или мутированную форму этого белка-мишени, где указанная экзогенная нуклеиновая кислота-мишень отличается от эндогенного гена-мишени на уровне нуклеиновой кислоты таким образом, что экспрессия этой экзогенной нуклеиновой кислоты-мишени существенно менее ингибируется двухцепочечной молекулой РНК, чем экспрессия эндогенного гена-мишени.
34. Клетка или организм по п.33, где экзогенная нуклеиновая кислота-мишень слита с дополнительной последовательностью нуклеиновой кислоты, кодирующей детектируемый пептид или полипептид.
35. Применение клетки или организма по любому из пп.30-34 для аналитических процедур.
36. Применение по п.35 для анализа профилей экспрессии генов.
37. Применение по п.35 для анализа протеома (белкового эквивалента генома).
38. Применение по любому из пп.35-37, где проводят анализ варианта или мутантной формы белка-мишени, кодируемого экзогенной нуклеиновой кислотой-мишенью.
39. Применение по п.38 для идентификации функциональных доменов белка-мишени.
40. Применение по любому из пп.35-39, где проводят сравнение по меньшей мере двух клеток или организмов, выбранных из
(i) контрольной клетки или контрольного организма без ингибирования гена-мишени,
(ii) клетки или организма с ингибированием клетки-мишени и
(iii) клетки или организма с ингибированием гена-мишени плюс комплементацией гена-мишени экзогенной нуклеиновой кислотой-мишенью.
41. Применение по любому из пп.35-40, где анализ включает в себя функциональный и/или фенотипический анализ.
42. Применение клетки по любому из пп.30-34 для препаративных процедур.
43. Применение по п.41 для выделения белков или белковых комплексов из эукариотических клеток.
44. Применение по п.43 для выделения высокомолекулярных белковых комплексов, которые могут необязательно содержать нуклеиновые кислоты.
45. Применение по любому из пп.35-44 в процедуре для идентификации и/или характеристики фармакологических агентов.
46. Система для идентификации и/или характеристики фармакологического агента, действующего по меньшей мере на один белок-мишень, содержащая
(а) эукариотическую клетку или эукариотический организм (не человека), способные экспрессировать по меньшей мере один ген-мишень, кодирующий указанный по меньшей мере один белок-мишень,
(b) по меньшей мере одну двухцепочечную молекулу РНК, способную ингибировать экспрессию указанного по меньшей мере одного эндогенного гена-мишени, и
(с) тест-вещество или коллекцию тест-веществ, где фармакологические свойства указанных тест-веществ или указанной коллекции подлежат идентификации и/или характеристике.
47. Система по п.46, дополнительно содержащая (d) по меньшей мере одну экзогенную нуклеиновую кислоту-мишень, кодирующую белок-мишень или вариант или мутированную форму этого белка-мишени, где указанная экзогенная нуклеиновая кислота-мишень отличается от эндогенного гена-мишени на уровне нуклеиновой кислоты таким образом, что экспрессия этой экзогенной нуклеиновой кислоты-мишени существенно менее ингибируется двухцепочечной молекулой РНК, чем экспрессия эндогенного гена-мишени.
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