vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ20.598vavilov-2483Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYНокаут гена shp-2 приводит к повышению CAR-опосредованной цитотоксичности NK-клеток линии YTshp-2 gene knockout upregulates CAR-driven cytotoxicity of YT NK cellsСубраковаВ. Г.SubrakovaV. G.

Новосибирск

Novosibirsk

subrakova.vera@gmail.comКулемзинС. В.KulemzinS. V.

Новосибирск

Novosibirsk

skulemzin@mcb.nsc.ruБеловежецТ. Н.BelovezhetsT. N.

Novosibirsk

ЧикаевА. Н.ChikaevA. N.

Новосибирск

Novosibirsk

ЧикаевН. А.ChikaevN. A.

Новосибирск

Novosibirsk

https://orcid.org/0000-0001-7788-2249КовальО. А.KovalO. A.

Новосибирск

Novosibirsk

https://orcid.org/0000-0003-2830-4236ГорчаковА. А.GorchakovA. A.

Novosibirsk

https://orcid.org/0000-0002-9184-4238ТаранинА. В.TaraninA. V.

Новосибирск

Novosibirsk

Институт молекулярной и клеточной биологии, Сибирское отделение Российской академии наук; Новосибирский национальный исследовательский государственный университетРоссияInstitute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State UniversityRussian FederationИнститут молекулярной и клеточной биологии, Сибирское отделение Российской академии наукРоссияInstitute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of SciencesRussian FederationИнститут молекулярной и клеточной биологии, Сибирское отделение Российской академии наук; Институт химической биологии и фундаментальной медицины, Сибирское отделение Российской академии наукРоссияInstitute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of SciencesRussian Federation2020180320202418086Copyright © Субракова В.Г., Кулемзин С.В., Беловежец Т.Н., Чикаев А.Н., Чикаев Н.А., Коваль О.А., Горчаков А.А., Таранин А.В., 20202020Субракова В.Г., Кулемзин С.В., Беловежец Т.Н., Чикаев А.Н., Чикаев Н.А., Коваль О.А., Горчаков А.А., Таранин А.В.Subrakova V.G., Kulemzin S.V., Belovezhets T.N., Chikaev A.N., Chikaev N.A., Koval O.A., Gorchakov A.A., Taranin A.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/2483

Одна из самых распространенных причин смертей пациентов в России, наряду с болезнями системы кровообращения, - это онкологические заболевания. Перспективным средством в борьбе с раковыми клетками представляются NK-клетки (естественные киллеры), однако для успешного применения в терапии онкологических заболеваний необходимо обеспечить их накопление в опухолевых очагах, устойчивость к иммуносупрессивному микроокружению, а также более высокую цитотоксическую активность. Известно, что NK-лимфоциты уничтожают раковые клетки, экспрессирующие специфические стресс-лиганды; при этом баланс сигналов от ингибирующих и активирующих рецепторов на поверхности NK-клетки определяет, будет ли запущена цитотоксическая реакция. Один из теоретически возможных способов повышения цитотоксичности состоит в том, чтобы при помощи генетического редактирования усилить активационные сигнальные каскады в NK-клетках и/или ослабить ингибирующие, таким образом сместив баланс сигналов в сторону активации лимфоцитов и лизиса мишеней. NK-клетки с таким модифицированным цитотоксическим потенциалом могут эффективнее уничтожать раковые мишени, обладающие так называемой устойчивостью к лизису. В этой работе мы предлагаем дважды модифицировать NK-клетки. Во-первых, при помощи системы CRISPR/Cas9 проводить нокаутирование гена shp-2 (PTPN11), кодирующего белок Shp-2 - негативный регулятор активации NK-клеток; во-вторых, при помощи лентивирусных векторов интегрировать кассету, кодирующую CAR (химерный антигенный рецептор), способный специфично связываться с антигенами на поверхности раковой мишени и генерировать активирующий сигнал. В качестве модельной NK-клеточной линии нами была выбрана перевиваемая линия NK-подобного фенотипа YT, поскольку эти клетки не нуждаются в специфических цитокинах для культивирования и могут проявлять перфорин/гранзим-опосредованную цитотоксическую активность. Мы показали, что сочетание нокаута гена shp-2 и экспрессии CAR повышает цитотоксичность эффекторных клеток на модели NK-устойчивой клеточной линии аденокарциномы простаты человека Du-145, экспрессирующей специфично узнаваемый CAR антиген, белок PSMA. Подобные линии с «усиленным» цитотоксическим фенотипом в перспективе могут быть использованы для нужд противораковой терапии.

In Russia, cancer is the second leading cause of death following cardiovascular diseases. Adoptive transfer of NK cells is a promising approach to fight cancer; however, for their successful use in cancer treatment, it is necessary to ensure their robust accumulation at tumor foci, provide resistance to the immunosuppressive tumor microenvironment, and to engineer them with higher cytotoxic activity. NK lymphocytes are known to kill cancer cells expressing a number of stress ligands; and the balance of signals from inhibitory and activating receptors on the surface of the NK cell determines whether a cytotoxic reaction is triggered. We hypothesized that stronger cytotoxicity of NK cells could be achieved via gene editing aimed at enhancing the activating signaling cascades and/or weakening the inhibitory ones, thereby shifting the balance of signals towards NK cell activation and target cell lysis. Here, we took advantage of the CRISPR/Cas9 system to introduce mutations in the coding sequence of the shp-2 (PTPN11) gene encoding the signaling molecule of inhibitory pathways in NK cells. These shp-2 knock-out NK cells were additionally transduced to express a chimeric antigen receptor (CAR) that selectively recognized the antigen of interest on the target cell surface and generated an activating signal. We demonstrate that the combination of shp-2 gene knockout and CAR expression increases the cytotoxicity of effector NK-like YT cells against human prostate cancer cell line Du-145 with ectopic expression of PSMA protein, which is specifically targeted by the CAR.

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