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Vol 56(2022) N 5 p. 770-779; DOI 10.1134/S0026893322050156 Full Text

G.M. Yusubalieva1,2, E.B. Dashinimaev3, A.A. Gorchakov4, S.V. Kulemzin4, O.A. Brovkina1, A.A. Kalinkin1, A.G. Vinokurov1, M.V. Shirmanova4,5, V.P. Baklaushev1,2*

Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures

1Federal Research and Clinical Center of the Federal Medical and Biological Agency of Russia, Moscow, 115682 Russia
2Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
3Pirogov Russian National Research Medical University, Moscow, 117437 Russia
4Institute of Molecular and Cellular Biology, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia
5Volga National Research Medical University, Nizhny Novgorod, 603005 Russia

*Baklaushev.vp@fnkc-fmba.ru
Received - 2021-11-14; Revised - 2022-03-03; Accepted - 2022-03-30

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out β2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1+ overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH-/- or B2M-/- knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH-/- has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1+-CISH-/- and YT-VAV1+B2M-/- lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1+ line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

glioblastoma multiforme, NK cells, YT, CISH, β2-microglobulin, adoptive immunotherapy



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