biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2021-21-4-266-278biopreparat-354Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESСравнение различных технологий получения рекомбинантного аденоассоциированного вируса в лабораторном масштабеComparison of different technologies for producing recombinant adeno-associated virus on a laboratory scalehttps://orcid.org/0000-0002-2687-5185РябоваЕ. И.RyabovaE. I.

Рябова Екатерина Игоревна

ул. Гамалеи, д. 18, Москва, 123098

Ekaterina I. Ryabova

18 Gamaleya St., Moscow 123098

ryabovaei96@gmail.comhttps://orcid.org/0000-0003-3776-3856ДеркаевА. А.DerkaevA. A.

Деркаев Артем Алексеевич

ул. Гамалеи, д. 18, Москва, 123098

Artem A. Derkaev

18 Gamaleya St., Moscow 123098

derkaev.a@yandex.ruhttps://orcid.org/0000-0002-2063-2449ЕсмагамбетовИ. Б.EsmagambetovI. B.

Есмагамбетов Ильяс Булатович, кандидат биологических наук

ул. Гамалеи, д. 18, Москва, 123098

Ilias B. Esmagambetov, Cand. Sci. (Biol.)

18 Gamaleya St., Moscow 123098

esmagambetovib@gmail.comhttps://orcid.org/0000-0002-1289-3411ЩебляковД. В.ShcheblyakovD. V.

Щебляков Дмитрий Викторович, кандидат биологических наук

ул. Гамалеи, д. 18, Москва, 123098

Dmitry V. Shcheblyakov, Cand. Sci. (Biol.)

18 Gamaleya St., Moscow 123098

sdmitryv@yahoo.comhttps://orcid.org/0000-0002-0017-7784ДовгийМ. А.DovgiyM. A.

Довгий Михаил Андреевич

ул. Гамалеи, д. 18, Москва, 123098

Mikhail A. Dovgiy

18 Gamaleya St., Moscow 123098

imhail@yandex.ruhttps://orcid.org/0000-0003-3971-5823БырихинаД. В.ByrikhinaD. V.

Бырихина Дарья Валерьевна

ул. Гамалеи, д. 18, Москва, 123098

Daria V. Byrikhina

18 Gamaleya St., Moscow 123098

byrikhina@yandex.ruhttps://orcid.org/0000-0002-4130-177XПрокофьевВ. В.ProkofievV. V.

Прокофьев Владимир Владимирович

ул. Гамалеи, д. 18, Москва, 123098

Vladimir V. Prokofiev

18 Gamaleya St., Moscow 123098

vladimir.prokofev2609@gmail.comhttps://orcid.org/0000-0003-4525-0364ЧемодановаИ. П.ChemodanovaI. P.

Чемоданова Ирина Петровна

ул. Гамалеи, д. 18, Москва, 123098

Irina P. Chemodanova

18 Gamaleya St., Moscow 123098

irina4ip@yandex.ruФедеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи» Министерства здравоохранения Российской ФедерацииРоссияNational Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. GamaleyaRussian FederationФедеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф.Гамалеи» Министерства здравоохранения Российской ФедерацииРоссияNational Research Center for Epidemiology and Microbiology named after Honorary Academician N. F. GamaleyaRussian Federation202116112021214266278Copyright © Рябова Е.И., Деркаев А.А., Есмагамбетов И.Б., Щебляков Д.В., Довгий М.А., Бырихина Д.В., Прокофьев В.В., Чемоданова И.П., 20212021Рябова Е.И., Деркаев А.А., Есмагамбетов И.Б., Щебляков Д.В., Довгий М.А., Бырихина Д.В., Прокофьев В.В., Чемоданова И.П.Ryabova E.I., Derkaev A.A., Esmagambetov I.B., Shcheblyakov D.V., Dovgiy M.A., Byrikhina D.V., Prokofiev V.V., Chemodanova I.P.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/354

Векторы на основе аденоассоциированного вируса являются одними из наиболее перспективных для доставки трансгенов в различные органы и ткани. Рекомбинантный аденоассоциированный вирус (rAAV) способен трансдуцировать как делящиеся, так и неделящиеся клетки, обладает низкой иммуногенностью и способен обеспечивать долгосрочную экспрессию трансгенов. На сегодняшний день существуют технологии, позволяющие получать rAAV для применения in vivo, однако они не лишены недостатков, связанных с трудоемкостью, сложностями масштабирования и высокой стоимостью, поэтому вопрос об усовершенствовании технологических схем получения rAAV является актуальным. Цель работы: сравнение технологических подходов к получению rAAV, основанных на различных условиях культивирования трансфицированной клеточной линии HEK293 в лабораторном масштабе. Материалы и методы: в исследовании использовали культуру клеток HEK293, плазмидную систему AAV-DJ Packaging System, систему PlasmidSelect Xtra Starter Kit. В качестве модели для сравнения технологий использовали вектор rAAV с трансгеном однодоменного антитела, слитого с Fc-фрагментом IgG1, специфичного к ботулотоксину. Применяли метод трансфекции клеток HEK293 суперскрученной плазмидной ДНК, выделенной при помощи трехступенчатой хроматографической очистки. Определение подлинности препарата rAAV проводили методами электрофореза, иммуноблоттинга и полимеразной цепной реакции в режиме реального времени. Результаты: продемонстрирована эффективность получения суперскрученной формы плазмидной ДНК, применимой для эффективной трансфекции с целью получения rAAV. Проведено сравнение процесса транзиентной трансфекции и культивирования трансфицированных клеток HEK293 в условиях суспензии в колбах, адгезии в культуральных флаконах и адгезии в биореакторе BioBLU 5p на матрице из дисков Fibra-Cel с целью продукции rAAV. Выводы: показана возможность применения описанных подходов к очистке плазмидной ДНК, трансфекции и культивированию трансфицированных клеток в различных условиях для получения препарата rAAV, эспрессирующего ген антитела. Реактор BioBLU 5p с дисками Fibra-Cel был впервые использован для получения препаративных количеств rAAV в лабораторном масштабе, что позволило увеличить площадь поверхности адгезии при культивировании и трансфекции клеток и, как следствие, увеличить выход целевого продукта.

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

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