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RU2017142352A - Изменение популяций микроорганизмов и модификация микробиоты - Google Patents

Изменение популяций микроорганизмов и модификация микробиоты Download PDF

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RU2017142352A
RU2017142352A RU2017142352A RU2017142352A RU2017142352A RU 2017142352 A RU2017142352 A RU 2017142352A RU 2017142352 A RU2017142352 A RU 2017142352A RU 2017142352 A RU2017142352 A RU 2017142352A RU 2017142352 A RU2017142352 A RU 2017142352A
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bacteria
species
host cell
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RU2017142352A
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Джаспер КЛУБЕ
Мортен Соммер
Кристиан ГРОНДАЛЬ
ДЕР ХЕЛМ Эрик ВАН
Рубен ВАСКЕС-УРИБЕ
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Снипр Текнолоджиз Лимитед
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Priority claimed from GBGB1507773.8A external-priority patent/GB201507773D0/en
Priority claimed from GBGB1507776.1A external-priority patent/GB201507776D0/en
Priority claimed from GBGB1507774.6A external-priority patent/GB201507774D0/en
Priority claimed from GBGB1507775.3A external-priority patent/GB201507775D0/en
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Priority claimed from GBGB1509366.9A external-priority patent/GB201509366D0/en
Priority claimed from GBGB1510891.3A external-priority patent/GB201510891D0/en
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Priority claimed from GB1600417.8A external-priority patent/GB2531454A/en
Priority claimed from GBGB1600418.6A external-priority patent/GB201600418D0/en
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Claims (44)

1. Модифицирующая хозяина (HM) система CRISPR/Cas для модификации нуклеотидной последовательности-мишени бактериальной клетки-хозяина, где система содержит компоненты по (i)-(iv):-
(i) по меньшей мере одна последовательность нуклеиновой кислоты, кодирующая нуклеазу Cas, которая является эндогенной для клетки-хозяина;
(ii) последовательность-мишень клетки-хозяина и сконструированный модифицирующей хозяина (HM) массив CRISPR, содержащий спейсерную последовательность (HM-спейсер) и повторы, кодирующие HM-crРНК, где HM-crРНК содержит последовательность, которая способна гибридизоваться с последовательностью-мишенью хозяина для направления указанной Cas на мишень в клетке-хозяине для модификации последовательности-мишени;
(iii) не обязательно, последовательность tracrРНК или последовательность ДНК для экспрессии последовательности tracrРНК;
(iv) где указанные компоненты системы разделены между клеткой-хозяином и по меньшей мере одним вектором на основе нуклеиновой кислоты, который может трансформировать клетку-хозяина, в результате чего HM-crРНК направляет Cas на мишень для модификации последовательности-мишени в клетке-хозяине,
где каждый вектор лишен последовательности, кодирующей нуклеазу Cas; и
где каждый вектор представляет собой фаговый вектор
2. Система по п.1, где система предназначена для медицинского применения.
3. Применение модифицирующей хозяина (HM) системы CRISPR/Cas для изменения относительного соотношения субпопуляций первых и вторых бактерий в смешанной популяции бактерий ex vivo в окружающей среде, где вторые бактерии содержат клетки-хозяева,
где для каждой клетки-хозяина система содержит компоненты по (i)-(iv):-
(i) по меньшей мере одна последовательность нуклеиновой кислоты, кодирующая нуклеазу Cas, которая является эндогенной для клетки-хозяина;
(ii) последовательность-мишень клетки-хозяина и сконструированный модифицирующей хозяина (HM) массив CRISPR, содержащий спейсерную последовательность (HM-спейсер) и повторы, кодирующие HM-crРНК, где HM-crРНК содержит последовательность, которая способна гибридизоваться с последовательностью-мишенью клетки-хозяина для направления указанной Cas на мишень в клетке-хозяине для модификации последовательности-мишени;
(iii) не обязательно, последовательность tracrРНК или последовательность ДНК для экспрессии последовательности tracrРНК;
(iv) где указанные компоненты системы разделены между клеткой-хозяином и по меньшей мере одним вектором на основе нуклеиновой кислоты, который трансформирует клетку-хозяина, в результате чего HM-crРНК направляет Cas на мишень для модификации последовательности-мишени в клетке-хозяине;
где последовательность-мишень модифицируют посредством Cas, в результате чего клетку-хозяина уничтожают, или рост клетки-хозяина уменьшают;
где каждый вектор лишен последовательности, кодирующей нуклеазу Cas и
где каждый вектор представляет собой фаг.
4. Применение по п. 3 для обработки медицинской жидкости, поверхности, устройства или контейнера в промышленности или ex vivo; или для обработки водных путей, воды, напитка, пищевого продукта или косметического продукта, где клетка-хозяин(клетки-хозяева) содержатся в жидкости, поверхности, устройстве, контейнере, водных путях, воде, напитке, пищевом продукте или косметическом продукте, или на них.
5. Применение по п. 3, где в результате применения получают бактериальные культуры для введения человеку или животным для обеспечения преимущества для Bacteroidetes, которые являются комменсальными или симбиотическими для человека или животных.
6. Применение по п. 3 или 5, где смешанная популяция представляет собой популяцию микробиоты кишечника ex vivo; не обязательно, где клетки-хозяева представляют собой клетки Firmicutes или Clostridium dificle.
7. Применение или система по любому из предшествующих пунктов, где
(a)каждая клетка-хозяин принадлежит к штамму или виду, обнаруженному в микробиоте человека;(b) каждая клетка-хозяин представляет собой клетку-хозяина Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio или Clostridium;
(c) первые бактерии являются пробиотическими, комменсальными или симбиотическими для человека (например, в кишечнике человека);
(d) как первые, так и вторые бактерии представляют собой Firmicutes и представляют собой бактерии из различных видов или штаммов;
(e) первые бактерии представляют собой Enterobacteriaceae, и вторые бактерии представляют собой Firmicutes; и
(f) клетки-хозяева представляют собой клетки архей вместо бактериальных клеток, или каждая популяция представляет собой популяцию архей вместо популяции бактерий.
8. Применение по любому из пп.3-7 для (a) изменения доли бактерий Bacteroidetes в смешанной популяции бактерий; (b) уменьшения доли субпопуляции Firmicutes (клеток-хозяев) в смешанной популяции бактерий; (c) уменьшения доли первого вида Firmicutes (клеток-хозяев) в смешанной популяции, где смешанная популяция содержит второй вид Firmicutes, рост которого не ингибируют посредством указанной cРНК; (d) уменьшения доли первого грамположительного вида бактерий (клеток-хозяев) в смешанной популяции бактерий, где смешанная популяция содержит второй грамположительный вид бактерий, рост которого не ингибируют посредством указанной cРНК; (e) уменьшения доли вида бактерий (клеток-хозяев) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, рост которого не ингибируют посредством указанной cРНК, где первый вид обладает последовательностью ДНК, кодирующей 16s рибосомальную РНК, которая является по меньшей мере на 80% идентичной последовательности ДНК, кодирующей 16s рибосомальную РНК другого вида; (f) уменьшения доли первого вида бактерий микробиоты кишечника человека (клеток-хозяев, например, Firmicutes) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, где другой вид представляет собой пробиотический вид кишечника человека, рост которого не ингибируют посредством указанной cРНК; или (g) уменьшения доли вида бактерий микробиоты кишечника человека ((клеток-хозяев, например, Firmicutes) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, где другой вид представляет собой вид, комменсальный для кишечника человека, рост которого не ингибируют посредством указанной cРНК.
9. Система по любому из пп.1,2 и 7 для (a) изменения доли бактерий Bacteroidetes в смешанной популяции бактерий; (b) уменьшения доли субпопуляции Firmicutes (клеток-хозяев) в смешанной популяции бактерий; (c) уменьшения доли первого вида Firmicutes (клеток-хозяев) в смешанной популяции, где смешанная популяция содержит второй вид Firmicutes, рост которого не ингибируют посредством указанной cРНК; (d) уменьшения доли первого грамположительного вида бактерий (клеток-хозяев) в смешанной популяции бактерий, где смешанная популяция содержит второй грамположительный вид бактерий, рост которого не ингибируют посредством указанной cРНК; (e) уменьшения доли вида бактерий (клеток-хозяев) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, рост которого не ингибируют посредством указанной cРНК, где первый вид обладает последовательностью ДНК, кодирующей 16s рибосомальную РНК, которая является по меньшей мере на 80% идентичной последовательности ДНК, кодирующей 16s рибосомальную РНК другого вида; (f) уменьшения доли первого вида бактерий микробиоты кишечника человека (клеток-хозяев, например, Firmicutes) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, где другой вид представляет собой пробиотический вид из кишечника человека, рост которого не ингибируют посредством указанной cРНК; или (g) уменьшения доли вида бактерий микробиоты кишечника человека (клеток-хозяев, например, Firmicutes) в смешанной популяции бактерий, где смешанная популяция содержит другой вид бактерий, где другой вид представляет собой вид, комменсальный для кишечника человека, рост которого не ингибируют посредством указанной cРНК; где (a)-(g) предназначены для лечения или предотвращения у субъекта - человека или животного (i) инфекции микробиоты указанным видом бактерий, долю которого уменьшают; или (ii) заболевания или состояния, опосредованного указанным видом бактерий, долю которого уменьшают.
10. Применение или система по любому из предшествующих пунктов для
(a) изменения относительного соотношения первой и второй субпопуляций в смешанной популяции бактерий, где вторые бактерии представляют собой Firmicutes;
(b) увеличения относительного соотношения Bacteroidetes к Firmicutes; или
(c) изменения доли бактерий Bacteroidetes в смешанной популяции бактерий
11. Применение или система по любому из предшествующих пунктов, где альтернативно, HM-crРНК и tracrРНК содержатся в одиночной направляющей РНК (gРНК), например, предоставленной посредством вектора.
12. Сконструированный вектор на основе нуклеиновой кислоты для применения в системе по любому из пп. 1,2,7 и 9-11 по п.1 для модификации бактериальной клетки-хозяина содержащей эндогенную систему CRISPR/Cas, где вектор представляет собой фаговый вектор,
(a) содержащий последовательности нуклеиновой кислоты для экспрессии множества различных crРНК (например, содержащихся в gРНК) для применения в системе CRISPR/Cas или для применения по любому из предшествующих пп.; и
(b) лишенный последовательности нуклеиновой кислоты, кодирующей нуклеазу Cas,
где первая из указанных crРНК является способной гибридизоваться с первой последовательностью нуклеиновой кислоты в указанной клетке-хозяине; и вторая из указанных crРНК является способной гибридизоваться с второй последовательностью нуклеиновой кислоты в указанной клетке-хозяине, где указанная вторая последовательность отличается от указанной первой последовательности; и
(c) первая последовательность содержится в гене устойчивости к антибиотику (или его РНК), и вторая последовательность содержится в гене устойчивости к антибиотику (или его РНК); необязательно, где гены являются различными;
(d) первая последовательность содержится в гене устойчивости к антибиотику (или его РНК), и вторая последовательность содержится в жизненно важном гене или гене вирулентности (или его РНК);
(e) первая последовательность содержится в жизненно важном гене (или его РНК), и вторая последовательность содержится в жизненно важном гене или гене вирулентности (или его РНК); или
(f) первая последовательность содержится в гене вирулентности (или его РНК), и вторая последовательность содержится в жизненно важном гене или гене вирулентности (или его РНК).
13. Применение, система или вектор по любому из предшествующих пунктов, где рост популяции клеток-хозяев уменьшают по меньшей мере в 5 раз по сравнению с ростом популяции указанных клеток-хозяев, не трансформированных указанным HM-массивом или нуклеотидной последовательностью, кодирующей указанную gРНК.
14. Применение, система или вектор по любому из предшествующих пунктов, где рост популяции клеток-хозяев на поверхности ингибируют.
15. Вектор на основе нуклеиновой кислоты в соответствии с вектором по любому из пп.12-14 или для использования в применении или системе по любому из пп.1-11,13 и 14, где вектор содержит более 1,4 т.п.о. экзогенной последовательности ДНК, где экзогенная ДНК кодирует один или несколько компонентов системы CRISPR/Cas и содержит сконструированный массив для экспрессии HM-crРНК или gРНК в клетках-хозяевах, где экзогенная последовательность лишена нуклеотидной последовательности, кодирующей нуклеазу Cas, родственную cРНК(нескольким cРНК) или gРНК(нескольким gРНК); где по меньшей мере 2 различные cРНК или gРНК кодированы экзогенной ДНК (например, посредством по меньшей мере 2 HM-массивов CRISPR).
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