Downregulation of TdT Expression through Splicing Modulation by Antisense Peptide Nucleic Acid (PNA)

Abstract

Background and Objective: Antisense oligonucleotides are able to modulate splicing patterns and offer therapeutic intervention for cancer and other diseases. Considering TdT potential as a target in cancer therapy, the present study aimed to investigate splicing alteration of TdT pre-mRNA in Molt-4 cells using peptide nucleic acid (PNA) octaarginine and cholic acid conjugates. Method: We examined 16 mer PNAs targeting 5' and 3' junctions of intron 7 and addressed their mRNA splicing modulation effects using RT-PCR analysis. We also tested corresponding 2-base mismatch PNAs to confirm the sequence specificity. In addition, protien level of TdT, apoptosis induction and cell viability rate were analysed. Results: PCR analysis showed that full match PNAs could modulate the splicing process, thereby producing a longer mRNA still including intron 7. PCR results also implied exon 7 skipping. In addition, reduced level of TdT protein in Molt-4 cells was observed. Downregulation of TdT level in PNA treated cells was accompanied by an increased rate of apoptosis and decreased the level of cell survival. Conclusion: PNA-mediated splicing modulation can specifically downregulate TdT expression. TdT dowregulation results in apoptosis induction and reduced cell survival in Molt-4 cells. These observations could draw more attentions to develop PNA based strategies for TdT suppression and consequent apoptosis induction in acute lymphoblastic leukemia.