ISSN 1866-8836
Клеточная терапия и трансплантация

GC-03. Lentiviral transduction efficiency of natural killer cells

Anastasia S. Mukhametshyna, Alexander A. Migas, Tatsiana V. Shman

Republican Research and Practical Center for Pediatric Oncology, Hematology and Immunology, Minsk, Republic of Belarus

Contact: Dr. Anastasia S. Mukhametshyna, phone: +375 (291) 752-676, e-mail:

doi 10.18620/ctt-1866-8836-2022-11-3-1-132


The active use of genetically modified natural killer (NK)-cells is limited by methodological difficulties of delivering genetic material to these cells. Lentiviral transduction is one of the cell modification methods. However, its use is limited by low yield of transduced cells. The virus derived from the VSV-G vector cassette, typically used to produce CAR-T cells, doesn’t efficiently transduce NK-cells. To increase the efficiency of NK-cell transduction, we studied alternative pseudotyped proteins. The pseudotyped lentiviral baboon envelope vector BaEV-TR shows higher transduction efficiency. It binds the entry receptors of the sodium-dependent neutral amino acid transporter ASCT-1 and ASCT-2 and is widely expressed in hematopoietic lineages. Thus, the use of alternative pseudotypes of lentiviruses will provide solution of many fundamental issues and specify the tools of obtaining sufficient numbers of genetically modified NK cells. Our objective was to compare the efficiency of NK-cell transduction using two variants of lentiviral particles based on the BaEV-TR and VSV-G vector cassettes.

Materials and methods

We used peripheral blood mononuclear cells from two healthy donors. A primary culture of NK-cells was obtained using a CD3-depletion kit, after which the cells were cultured in PRMI-1640 complete growth medium supplemented with IL-2 500 IU, 72 hours before transduction; two lentiviral vectors with different envelope plasmids were also used; in the first variant, pMD2.G was used, encoding the surface glycoprotein G of the vesicular stomatitis virus (VSV-G); in the second, BaEV-TR, encoding the modified envelope glycoprotein of the baboon retrovirus. The psPAX2 plasmids, containing gag-pol and pUltra genes which encode the EGFP reporter protein gene, were included in both variants. NK cells were grown in complete RPMI medium prior to transduction. The multiplicity of infection was of 1, 5, 10. After transduction of the virus, the cells were incubated at 37°C, and the medium was changed after 24 hours. The level of transduction was determined by flow cytometry by the expression of the EGFP reporter protein after 72 hours.

Results and discussion

We compared the transduction efficiency of primary NK cells with two variants of lentiviral particles based on the BaEV-TR and VSV-G vector cassettes. The percentages of transduced cells at the infection multiplicity of 1, 5, 10 with VSV-G and BaEV-TR were 0.28%, 1.5%, 2.8% and 50%, 62%, 70%, respectively. The VSV-G receptor is a low-density lipid receptor LDL-R which is not expressed in either resting or activated NK-cells [1] thus explaining the low transduction rate we obtained. However, the baboon envelope receptors (ASCT-1 and ASCT-2) are overexpressed in response to activation of NK-cells by IL-2, IL-12 and IL-21, thus leading to increased efficiency of BaEV-TR transduction [Bari et al., 2019] as confirmed by our results.


The percentage of NK cells transduced with a lentiviral vector employing BaEV-TR vector cassette is higher than with a VSV-G-based vector cassette, which may be further used for immunotherapy with modified NK cells.


Bari R [et al.] (2019) A distinct subset of highly proliferative and lentiviral vector (LV)-transducible NK-cells define a readily engineered subset for adoptive cellular therapy. Front. Immunol. 10:2001. doi:10.3389/fimmu.2019.02001


NK cells, transduction, lentivirus, pseudotyped envelope particles, retrovirus, baboon.

Volume 11, Number 3

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doi 10.18620/ctt-1866-8836-2022-11-3-1-132

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