Introduction
No cure, vaccine, or treatment is currently available for the ~10 million people worldwide living with Human T-cell Leukaemia Virus Type-1 (HTLV-1). Infection is lifelong and causes Adult T-cell Leukaemia/Lymphoma (ATLL) and HTLV-1 associated myelopathies (HAM). RNA therapeutics offer sequence specific targeting of HTLV-1. We designed and screened custom siRNA targeting the HTLV-1 promoters and oncogenes responsible for disease.
Materials/ Methods
Preliminary screens were performed in HTLV-1 293T-GFP transduced reporter cell lines. Cells were transfected with siRNAs using RNAiMax. GFP suppression was measured at day four using flow cytometry. Subsequent validation performed in MJ T-cell lymphoma cell line. Hbz and 3’LTR siRNA hits were validated on the mRNA level using RT-qPCR. Apoptosis was assessed by flow cytometry measuring Annexin V/ propidium iodide (Thermofisher Scientific).
Results
Significant GFP suppression observed in four 5’LTR, seven 3’LTR, seven tax, and fourteen hbz targeted siRNAs (p<0.05, n = 6) in the GFP reporter system after 96 hours. 13 novel anti-hbz and two anti-3’LTR promoter siRNAs demonstrated significant hbz mRNA knockdown and two novel promoter targeted siRNAs in MJ T cells (p<0.05, n = 3). Currently confirming anti-hbz siRNAs for induced apoptosis. We are currently examining the off target effects of 3'LTR and hbz targeted siRNAs, their dose response, and confirmation by Western Blot.
Conclusions
siRNAs can provide direct acting antiviral treatment by suppressing the activity of promoters and oncogenes. Confirmation on the protein level using Western Blots will provide further validation. Development of lipid nanoparticles delivery may provide the opportunity for clinical translation.