medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2023.11.20-26medgen-2370Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHРедактирование мутации c.3846G>A (p.Trp1282*) в гене CFTR в ИПСК с использованием аденинового редактораEditing the c.3846G>A (p.Trp1282*) mutation in the CFTR gene in iPSCs using adenine editorКондратьеваЕ. В.KondratevaE. V.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechie st., Moscow, 115522

ДемченкоА. Г.DemchenkoA. G.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechie st., Moscow, 115522

ЛавровА. В.LavrovA. V.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechie st., Moscow, 115522

СмирнихинаС. А.SmirnikhinaS. A.

115522, г. Москва, ул. Москворечье, д. 1

1, Moskvorechie st., Moscow, 115522

ФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»РоссияResearch Centre for Medical GeneticsRussian Federation20230812202322112026Copyright © Кондратьева Е.В., Демченко А.Г., Лавров А.В., Смирнихина С.А., 20232023Кондратьева Е.В., Демченко А.Г., Лавров А.В., Смирнихина С.А.Kondrateva E.V., Demchenko A.G., Lavrov A.V., Smirnikhina S.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.medgen-journal.ru/jour/article/view/2370

Муковисцидоз (МВ) – аутосомно-рецессивное заболевание, обусловленное мутациями в гене CFTR, приводящими к дисбалансу ионов хлора и натрия в эпителиальных клетках различных органов. Нонсенс-мутации в гене CFTR описаны у 10% пациентов с МВ, при этом наиболее частой нонсенс-мутацией является c.3846G>A (p.Trp1282*, W1282X). Известно, что современная патогенетическая терапия МВ CFTR-модуляторами не эффективна в отношении этого класса мутаций, поэтому пациенты с нонсенс- мутациями в гене CFTR до сих пор остаются без эффективного лечения. Этиотропная терапия МВ может быть разработана на основе новейших методов геномного редактирования, например, с использованием редакторов оснований, позволяющих точечно изменять нуклеотиды в геноме. Адениновый редактор оснований позволяет целенаправленно корректировать нонсенс- мутации. Целью работы явилась оценка эффективности коррекции мутации c.3846G>A в индуцированных плюрипотентных стволовых клетках (ИПСК) пациента с МВ с помощью аденинового редактора оснований. В работе использован редактор xCas9(3.7)-ABE(7.10) в сочетании с гидовой РНК (в отдельной плазмиде B52-1282), позволяющей редактору конвертировать c.3846A>G. Плазмиды были трансфицированы электропорацией в ИПСК с генотипом F508del/c.3846G>A в гене CFTR. Оценку эффективности коррекции проводили спустя 48 часов путём глубокого таргетного секвенирования. Результаты работы показали, что частота конверсии нуклеотида c.3846А>G составила 10,9% аллелей. При этом частота нежелательных изменений (инделов) в локусе редактирования не превышала таковую в нетрансфицированном контроле. Таким образом, в работе показано, что адениновый редактор оснований xCas9(3.7)-ABE(7.10) позволяет корректировать мутацию c.3846G>A в гене CFTR в 10,9% аллелей в ИПСК больного МВ без внесения дополнительных мутаций в локус редактирования.

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, leading to an imbalance of chloride and sodium ions in the epithelial cells of various organs. Nonsense mutations in the CFTR gene are present in 10% of CF patients, with the most common being c.3846G>A (p.Trp1282*, W1282X). Current CFTR-modulator therapy is ineffective against this class of mutations, leaving patients with nonsense mutations in the CFTR gene without effective treatment. Etiotropic therapy for CF can be developed based on the latest genome editing methods, such as base editors, which allow precise changes to nucleotides in the genome. Adenine base editors allow for targeted correction of nonsense mutations. The aim of this study was to evaluate the effectiveness of correcting the c.3846G>A mutation in induced pluripotent stem cells (iPSCs) from a CF patient using an adenine base editor. The xCas9(3.7)-ABE(7.10) editor was used in combination with a guide RNA (in a separate B52-1282 plasmid) to convert c.3846A>G. Plasmids were transfected into iPSCs with the F508del/c.3846G>A genotype in the CFTR gene using electroporation. The effectiveness of correction was evaluated 48 hours later using deep targeted sequencing. The results showed a conversion frequency of 10.9% of c.3846A>G alleles, with no increase in unwanted changes (indels) in the editing locus compared to the untransfected control. Therefore, this study demonstrates that the adenine base editor xCas9(3.7)-ABE(7.10) allows for correction of the c.3846G>A mutation in the CFTR gene in 10.9% of alleles in iPSCs from a CF patient without introducing additional mutations in the editing locus.

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