Abstract PO055: A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides

Background: Current cancer treatment tactics ought to be selective to carcinogenic cells and non-destructive to normal cells. Telomerase is a ribonucleoprotein enzyme which plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. While it is not found in somatic cells, its reactivation is obligatory in malignant cells to maintain the length of the telomere and prevent its senescence. Antisense-based strategies against human Telomerase RNA component (hTR) led to cancer cell death at some time after administering of antisense oligonucleotide (ASOs), and this lag time is one of the major drawbacks for this approach in treating cancer. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation. CENP-B is highly expressed in cancer cells helping in high rate proliferation. Materials and Methods: HepG2 cells were transfected with several concentrations (6.25, 12.5 and 25 nM) of LNA ASO against hTR and CENP-B mRNA either individually or in combination. MTT assay was used to assess the Hep-G2 cytotoxicity 24 and 48 hrs post-transfection. Telomerase activity, hTR, and CENP-B mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Effect of antisense treatment on Caspase-3, Bax, and Bcl-2 was also investigated by RT-PCR. Transforming growth factor (TGF-β), Tumor necrosis factor (TNF-α) and, Nitric oxide (NO) were estimated in supernatant collected 48 after transfection with ASOs by enzyme-linked immunosorbent assay (ELISA). Results: Our study conclusively demonstrates that using a single treatment with LNA ASO- hTR or CENP-B led to dose and time-dependent reduction in HepG2 cell viability. The combination of two LNA antisense (hTR + CENP-B) with different concentrations showed a synergistic cytotoxic effect. LNA ASO treatments (6.25 nM from each) exhibit the greatest synergistic cytotoxic effect. hTR and CENP-B mRNA in HepG2 cells were totally abrogated. Telomerase activity was disappeared. Expression of Caspase-3, Bax, and Bcl-2 was not detected after transfecting cells with LNA ASO hTR alone or in-combination with LNA ASO CENP-B indicates to caspase-independent cell death. A significant decrease in TGF-β and TNF-α and elevation in NO secretions was observed. Conclusion: LNA ASO hTR is potent in decreasing telomerase activity with a rapid anti-tumor effect with Caspase-independent cell death. This tumor inhibitory effect was magnified by ASO CENP-B indicating to the synergistic cytotoxic effect of oligonucleotides combination at certain concentrations. The rapid inhibitory effects of combined oligonucleotides on tumor growth open a new prospective application of antisense in cancer therapy. Citation Format: Ahmed Mohamed El-Desoky, Yasser Ali, Roba Talaat. A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO055.