Functional Analysis of EBV-Derived microRNAs in Systemic Chronic Active EBV Disease Using Novel Antisense Oligonucleotides

Background and Aim

Systemic chronic active Epstein-Barr virus disease (sCAEBV) is one of the EBV-positive T- or NK-cell lymphoproliferative diseases. sCAEBV, which is accompanied by systemic inflammation, can lead to fatal disorders such as hemophagocytic lymphohistiocytosis (HLH). Understanding the pathogenesis that causes persistent inflammation in sCAEBV is an urgent issue. The EBV genome encodes 40 mature microRNAs known as BamHI-A region rightward transcript miRNAs (miR-BARTs). Although miR-BARTs have been reported to contribute to the development of B-cell lymphoma and nasopharyngeal carcinoma, the role of miR-BARTs in the pathogenesis of sCAEBV has never been elucidated. In our previous analyses, single knock down of miR-BART7-3p, the most prominently expressed miR-BART, did not present a clear impact on the EBV-infected cells of sCAEBV (ASH2022). In this study, therefore, we aimed to clarify the function of miR-BARTs and their potential as therapeutic targets of sCAEBV using our newly developed antisense oligonucleotides (ASOs) targeting all miR-BARTs.

Methods

sCAEBV was diagnosed according to the criteria which match the definition of the WHO classification of hematopoietic malignancies. We used EBV-positive T- (SNT15 and SNT16) and NK- (SNK1 and SNK10) cell lines established from sCAEBV patients (Br J Hem. 2003:21;805). EBV-infected cells were isolated from peripheral blood mononuclear cells of the patients with sCAEBV using magnetic beads. The expression of miR-BARTs was examined by miRNA-sequencing using HiSeq2000® and TaqMan® miRNA assays. Primary transcript of BART mRNA was identified using a nanopore sequencing. We developed novel ASOs targeting the BART primary transcript to silence overall expression of all miR-BARTs. The introduction of these ASOs into cells was carried out using Lonza Nucleofector® 2b. miRNA expression in the histopathological specimen was examined by dual in situ hybridization immunohistochemistry using miRNA scope™.

Results

In EBV-positive T- or NK-cell lines and EBV-infected T- or NK-cells from patients with sCAEBV, miRNA sequencing showed highly abundant expression of 40 miR-BARTs. In the plasma of patients with sCAEBV, there was also highly abundant expression of 40 miR-BARTs. It is commonly considered that miR-BARTs are produced through splicing from a primary transcript of BART gene. Nanopore sequencing detected mature BART mRNAs bearing a poly A tail. This fact suggests the presence of a primary transcript containing the full-length BART region. Our newly developed ASOs suppressed the expression of the BART primary transcript and all 40 miR-BARTs in EBV-positive T- or NK-cell lines. The more reactive cell lines exhibited higher inhibition of cell proliferation by ASOs. Gene ontology analysis demonstrated that the expression of apoptosis- and immune-response-related genes enhanced after the introduction of our ASOs. Additionally, we observed that EBV-infected T- or NK-cells secreted extracellular vesicles (EVs) containing miR-BARTs and that miR-BART7-3p was in macrophages from sCAEBV patients' lesions. ASO treatment markedly reduced miR-BARTs expression in these EVs.

Conclusion

Our ASOs successfully suppressed miR-BART expression comprehensively for the first time. In sCAEBV, multiple miR-BARTs may cooperate to regulate cell proliferation and immune evasion. miR-BARTs have a potential not only to inhibit the cell proliferation of EBV-infected cells but also to regulate the tumor microenvironment in sCAEBV.

Nabeshima:Ono Pharmaceutical Co., Lt: Current Employment. Arai:Sanofi K.K.: Honoraria; Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Honoraria, Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Eisai Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Bristol-Myers Squibb K.K.: Honoraria; AbbVie GK: Honoraria; Janssen Pharmaceutical K.K: Honoraria.