Cell and Tissue BiologyCell and Tissue BiologyЦитология0041-37713034-6061The Russian Academy of Sciences66950210.31857/S0041377124040064QCPXOWArticlesСтатьиResearch ArticleDevelopment of an in vitro model of dysferlinopathy via crispr/cas-mediated transcriptional activation of the dysf geneРазработка in vitro модели дисферлинопатии посредством crispr/cas-опосредованной активации гена dysfYakovlevI. A.ЯковлевИ. А.
Russian Federation
mail@genotarget.comSlesarenkoY. S.СлесаренкоЯ. С.
Russian Federation
mail@genotarget.comStarostinaI. G.СтаростинаИ. Г.
Russian Federation
mail@genotarget.comShaimardanovaA. A.ШаймардановаА. А.
Russian Federation
mail@genotarget.comSolovyovaV. V.СоловьеваВ. В.
Russian Federation
mail@genotarget.comBobrovskyP. A.БобровскийП. А.
Russian Federation
mail@genotarget.comGrafskaiaE. N.ГрафскаяЕ. Н.
Russian Federation
mail@genotarget.comBelikovaL. D.БеликоваЛ. Д.
Russian Federation
mail@genotarget.comBardakovS. N.БардаковС. Н.
Russian Federation
mail@genotarget.comRizvanovA. A.РизвановА. А.
Russian Federation
mail@genotarget.comIsaevA. A.ИсаевА. А.
Russian Federation
mail@genotarget.comDeevR. V.ДеевР. В.
Russian Federation
mail@genotarget.comArtgene BiotechПАО “Артген биотех”OOO Genotarget, Skolkovo Innovation CenterООО “Генотаргет”, Инновационный центр “Сколково”Kazan (Volga Region) Federal UniversityКазанский (Приволжский) федеральный университетLopukhin Federal Research and Clinical Center of Physical-Chemical MedicineФедеральный научно-клинический центр физико-химической медицины им. Ю. М. Лопухина ФМБА РоссииDivision of Medical and Biological Sciences, Tatarstan Academy of SciencesОтделение медицинских и биологических наук, АН Республики ТатарстанAvtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of SurgeryНаучно-исследовательский институт морфологии человека им. акад. А.П. Авцына1507202466438039227022025Copyright ©; 2024, Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024Russian Academy of SciencesРоссийская академия наукhttps://vietnamjournal.ru/0041-3771/article/view/669502

Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening in order to select the most effective approach to restore the synthesis of the target protein. We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.

Для разработки методов генной терапии и геномного редактирования при моногенных заболеваниях необходимы клеточные модели из тканей человека, полученных малоинвазивными методами, позволяющие провести скрининг и выбрать наиболее эффективный подход по восстановлению синтеза целевого белка. В работе применена система транскрипционной активации CRISPR/dCas9-SAM, обеспечивающая экспрессию гена DYSF в клетках линии HEK 293Т, а также в фибробластах десны пациента с дисферлинопатией (с гомозиготной мутацией c. 2779delG (Ala9 27LeufsX 2 1)). После активации гена DYSF удалось детектировать его функциональные продукты (мРНК гена и белок) в транскрипционно активированных (ТА) клетках HEK293Т (HEK293Т_ТА) и мРНК в фибробластах. Активация транскрипции интересующего гена в фибробластах и клеточной линии HEK 293Т_ТА может быть использована для in vitro оценки эффективности геномного редактирования и генной терапии дисферлинопатии. Активируя ген, участвующий в развитии той или иной патологии, можно впоследствии использовать системы редактирования генома, а также конструкции для генной терапии. Это позволит более точно изучать вклад различных мутаций в патогенез заболевания и разрабатывать этиотропное лечение.

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