Активность CAG-промотора при rAAV-опосредованной генной терапии: тканеспецифичность и возрастная динамика

ВВЕДЕНИЕ. Несмотря на широкое использование в генотерапевтических препаратах на основе рекомбинантных аденоассоциированных вирусов (rAAV) искусственных промоторов, основанных на промоторе β-актина курицы (обозначаемых как CAG, CBA или CB), данные об активности таких промоторов в разных тканях и возрастной динамике остаются ограниченными. В настоящей работе проведена количественная оценка активности CAG-промотора в различных органах мышей в постнатальном периоде для определения его применимости при разработке rAAV-опосредованной терапии.

ЦЕЛЬ. Оценка относительной активности CAG-промотора и ее динамики в органах мышей в возрасте 3–12 недель.

МАТЕРИАЛЫ И МЕТОДЫ. 2-дневным мышам линии ICR (CD-1) однократно вводили rAAV9 с кассетой для экспрессии SMN под контролем CAG-промотора. На 3, 6 и 12-й нед. после инъекции из образцов органов (головной мозг, спинной мозг, печень, легкие, сердце, четырехглавая мышца бедра) выделяли тотальную ДНК и РНК. Содержание вирусных геномов rAAV и мРНК SMN в препаратах нуклеиновых кислот определяли методом количественной ПЦР. Относительную активность промотора рассчитывали как отношение концентрации мРНК SMN к концентрации вирусных геномов (rAAV), нормализованное на соотношение РНК:ДНК в органе. Для анализа данных вычисляли среднее геометрическое и использовали регрессионный анализ.

РЕЗУЛЬТАТЫ. Активность CAG-промотора значительно варьировала в разных органах. На 3 нед. после инъекции наибольшие значения активности отмечены в четырехглавой мышце бедра, в 4,7–9,7 раза превышающие таковые в других органах. Также обнаружено, что в головном мозге и легких с 3 по 12 нед. после инъекции активность промотора снижалась в 4,7 раза (p=0,0094) и в 5,2 раза (p=0,0039) соответственно, в то время как в других тканях существенных изменений активности не наблюдалось.

ВЫВОДЫ. CAG-промотор малопригоден для экспрессии трансгена в клетках легких и головного мозга вследствие транскрипционного сайленсинга, а также не является оптимальным для экспрессии в клетках печени из-за относительно низкой активности. Эти данные следует учитывать при разработке генотерапевтических препаратов.

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