ВВЕДЕНИЕ

biopreparat

БИОпрепараты. Профилактика, диагностика, лечение

Biological Products. Prevention, Diagnosis, Treatment

2221-996X2619-1156

Scientific Centre for Expert Evaluation of Medicinal Products

10.30895/2221-996X-2026-26-2-159-170

biopreparat-774

Research Article

ТЕМА НОМЕРА: РАЗРАБОТКА ПРЕПАРАТОВ ДЛЯ ЛЕЧЕНИЯ РЕДКИХ (ОРФАННЫХ) ЗАБОЛЕВАНИЙ

ISSUE TOPIC: DEVELOPMENT OF MEDICINAL PRODUCTS FOR THE TREATMENT OF RARE (ORPHAN) DISEASES

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

Tissue specificity and age-related dynamics of CAG promoter activity in rAAV-mediated gene therapy

https://orcid.org/0009-0000-9131-3464

Анисимов

Р. Л.

Anisimov

R. L.

Анисимов Роман Львович, канд. биол. наук

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Roman L. Anisimov, Cand. Sci. (Biol.)

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

anisimov@ibcgenerium.ru

https://orcid.org/0009-0004-8151-5758

Никифорова

Н. В.

Nikiforova

N. V.

Никифорова Наталья Владимировна

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Natalya V. Nikiforova

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

https://orcid.org/0009-0005-0441-4918

Иванов

Е. С.

Ivanov

E. S.

Иванов Евгений Сергеевич

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Evgeny S. Ivanov

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

https://orcid.org/0000-0003-0628-5513

Овсепян

А. А.

Ovsepyan

A. A.

Овсепян Армен Александрович

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Armen A. Ovsepyan

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

https://orcid.org/0000-0002-9199-8464

Борзов

А. А.

Borzov

A. A.

Борзов Антон Александрович

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Anton A. Borzov

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

https://orcid.org/0000-0003-0682-6113

Казаров

А. А.

Kazarov

A. A.

Казаров Александр Александрович

Ул. Владимирская, д. 14, пос. Вольгинский, городской округ Покров, Владимирская область, 601125

Alexander A. Kazarov

14 Vladimirskaya St., Volginsky, Pokrov, Vladimir Region 601125

Акционерное общество «ГЕНЕРИУМ»РоссияGENERIUM JSCRussian Federation

2026

04072026

262159170

2026

Анисимов Р.Л., Никифорова Н.В., Иванов Е.С., Овсепян А.А., Борзов А.А., Казаров А.А.

Anisimov R.L., Nikiforova N.V., Ivanov E.S., Ovsepyan A.A., Borzov A.A., Kazarov A.A.

Данная работа распространяется под лицензией Creative Commons Attribution 4.0.

This work is licensed under a Creative Commons Attribution 4.0 License.

https://www.biopreparations.ru/jour/article/view/774

ВВЕДЕНИЕ

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

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

МАТЕРИАЛЫ И МЕТОДЫ

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

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

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.

INTRODUCTION

INTRODUCTION. Artificial promoters based on the chicken beta-actin promoter (CAG, CBA, or CB) are frequently used in recombinant adeno-associated virus (rAAV)-based gene therapy products. However, data on the activity and age-related activity dynamic of these promoters remain quite limited. In this study, we quantitatively assessed CAG promoter activity in various organs of postnatal mice to determine its suitability for the development of rAAV-mediated gene therapy.

AIM

AIM. Evaluation of the relative CAG promoter activity and its dynamics in mouse organs from 3 to 12 weeks of life.

MATERIALS AND METHODS

MATERIALS AND METHODS. Two-day-old ICR (CD-1) mice were injected once with rAAV9 carrying a cassette for SMN expression under the control of the CAG promoter. At 3, 6, and 12 weeks after administration, total DNA and RNA were isolated from organ samples (brain, spinal cord, liver, lungs, heart, quadriceps femoris muscle). The content of rAAV genomes and SMN mRNA in nucleic acid preparations were determined by quantitative PCR. Relative promoter activity was calculated as the ratio of the SMN mRNA concentration to the concentration of rAAV genomes, normalized on the RNA:DNA ratio in the organ. Data were analyzed using the geometric mean and regression analysis.

RESULTS

RESULTS. CAG promoter activity varies significantly in different organs. At week 3 after injection, the highest values were observed for the quadriceps femoris muscle, 4.7–9.7 times higher than those in other organs. It was also found that in the brain and lungs, promoter activity decreased 4.7-fold (p=0.0094) and 5.2-fold (p=0.0039), respectively, from 3 to 12 weeks after injection, while in other tissues no significant changes in activity were observed.

CONCLUSIONS

CONCLUSIONS. The CAG promoter is poorly suited for transgene expression in the lung and brain cells due to promoter silencing and is suboptimal for expression in the liver cells because of relatively low activity; these findings should be taken into account in the development of gene therapy products.

генная терапиярекомбинантные аденоассоциированные вирусыrAAVCAG-промоторспинальная мышечная атрофия

gene therapyrecombinant adeno-associated virusrAAVCAG promoterspinal muscular atrophy

Работа проводилась при финансировании АО «ГЕНЕРИУМ»

The work was funded by GENERIUM JSC

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The authors declare that there are no conflicts of interest present.