Background: No cure, vaccine, or treatment is currently available for the ~20 million people worldwide living with the lifelong infection caused by Human T-cell Leukaemia Virus Type-1 (HTLV-1). Infection results in Adult T cell Leukaemia (ATL), myelopathy (HAM), and other immune-associated diseases. HTLV-1 subtype A is the most prevalent global subtype, but the highly divergent subtype C is endemic to communities within Central Australia. RNA therapeutics offer sequence specific targeting of HTLV-1 subtypes. In this study, we developed and screened custom siRNA targeting the viral regions responsible for disease: HTLV-1 promoters 5’LTR, 3’LTR, and oncogenes tax and hbz. We demonstrated novel siRNAs induce potent silencing of viral mRNA and have potential to be multiplexed for broad spectrum coverage of global subtypes A and C.
Methods: Antiviral efficacy of novel, highly conserved siRNAs were assessed in T-cell lymphoma cell lines MJ and MT-2. Viral suppression mediated by 5’LTR, 3’LTR, tax and hbz targeted siRNAs were validated on the mRNA level using RT-qPCR. All siRNA were assessed for off target interferon stimulated gene (ISG) response induction in HeLA T4 or MT-2 cells, and its potency determined via dose response with log fold dilutions (5 nM, 0.5 nM, 0.05 nM, 0.005 nM). In silico identification of multiplexed siRNA candidates was determined using MultipLExed tARget sequeNce aNalysis (LEARNN) program against 351 full length genomes, including 29 HTLV-1c sequences. Lipofectamine RNAiMax was used to transfect all siRNA.
Results: Viral suppression was induced by 13 hbz-targeted, six 3’LTR, six tax and six 5’LTR siRNAs, which demonstrated significant hbz or tax mRNA knockdown in leukaemic T cells (p<0.05, n = 3). Of these hits, three 3’LTR and hbz targeted siRNAs demonstrated silencing potency down to 0.05 nM, and three tax targeted siRNAs and two 5’LTR targeted siRNAs suppressing potently at a 0.005 nM dose (p<0.05, n = 3). No significant off target effects were induced by siRNAs across four ISGs assessed. Potent siRNAs were assessed for multiplex potential, and 40 combinations identified. Multiplexing yields minimum global coverage of 99%, with 31 combinations providing 100% coverage, including all tax and hbz-targeted siRNA combinations.
Discussion and conclusions: This study demonstrates siRNAs can provide direct acting antiviral treatment by suppressing the activity of promoters and oncogenes in infected T cell lines. siRNAs have high multiplexing potential for broad spectrum coverage of global and Australian subtypes. Future development of siRNA using lipid nanoparticles may provide the opportunity for clinical translation.