HLA-G gene editing in a choriocarcinoma cell line as a novel tool in cancer therapies

PALMA MARÍA BELÉN; TRONIK-LE ROUX, DIANA; SEOANE ROCHA, CAMILA; IRIBARNE, AILEN; LA GRECA, ALEJANDRO; MORO, LUCÍA N.; CAROSELLA, EDGARDO D.; GARCÍA, MARCELA N.; MIRIUKA, SANTIAGO G.

Congreso; Reunión Anual de Sociedades de Biociencias; 2021

SAIC-SAI-AAFE-NANOMED

Cancer immunotherapies, based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies, offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to these therapies. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several tumor malignancies generating a tolerogenic microenvironment. Here, CRISPR/Cas9 gene editing system was used to block the HLA-G expression in a choriocarcinoma cell line named JEG-3, which expresses high levels of this protein. For this purpose, four different single guide-RNA targeting HLA-G exon 1 and 2 were designed (named 1A-, 1B-, 2A- and 2B-sgRNA). Then, these sgRNAs were cloned into pSpCas9(BB)-2A-puro(PX459) vector and were transfected in JEG-3 cells, each one separately or all four plasmids simultaneously. The HLA-G expression was measured in the edited JEG-3 cells by western blotting, flow cytometry and RT-qPCR. Genomic DNA was evaluated by Sanger sequencing to analyse possible modifications. The results showed that HLA-G gene editing was achieved. Downregulation of HLA-G was reached to different degrees when JEG-3 cells were edited with each one sgRNA separately. Whereas, complete HLA-G silencing was achieved when JEG-3 cells were edited with all sgRNAs simultaneously. Most importantly, the NK degranulation assay showed that edited JEG-3 cells (HLA-G negatives) triggered a higher in vitro response of NK cells with respect to wild type JEG-3 cells (HLA-G positives). Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing, with a concomitant effect in immune cell activation. This approach would reactivate the host immune system and help to eliminate tumor cells, thus proposing novel cancer immunotherapy.