| There has been a great deal of interest in harnessing the power of RNA interference (RNAi) for the treatment of human disease. RNAi is a process through which small dsRNA molecules known as short interfering RNAs (ds-siRNAs) targets homologous transcripts for degradation. One disease that may be amenable to an RNAi-based therapeutic is human papillomavirus (HPV) infection, the primary cause of cervical cancer. The oncogenic properties of HPV are due to the proliferative and transformative activities of the E6 and E7 oncoproteins. However, it was not known whether HPV oncogene expression was still required for continued cell growth after the initial transformation. To test this, we used RNA interference to inhibit expression of E6 and E7 in HeLa cells (an HPV 18-transformed cell line). Because E6 and E7 are expressed together on polycistronic mRNAs, we were able to use a single ds-siRNA molecule targeted to E7 to silence both genes. We found that this E7 ds-siRNA inhibited DNA synthesis, induced morphologic and biochemical changes characteristic of senescence and caused long term growth arrest, demonstrating that reducing E6 and E7 expression alone is sufficient to cause HeLa cells to become senescent. These results raise the possibility of using an anti-E6/E7 ds-siRNA to treat HPV-related disease. However, native ds-siRNA molecules were unlikely to be efficient therapeutic agents without protection from intracellular and extracellular nucleases. We tested several novel boranophosphate (BP) backbone ds-siRNAs for nuclease susceptibility and silencing activity. We found that peripherally-modified BP ds-siRNAs were consistently more potent and nuclease resistant than native molecules. The enhanced stability of BP ds-siRNA suggested another possible application: BP modification of short, single-stranded antisense RNA (ss-siRNA), which had recently been shown to induce RNA interference. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared to ds-siRNA, thought to be related to its susceptibility to nuclease degradation, has prevented its widespread use. We found that boranophosphate modification of ss-siRNA lead to enhanced nuclease resistance as well as improved and prolonged silencing, when compared to native single-stranded or double-stranded siRNA molecules. The potency and stability of BP ss-siRNAs indicates that they merit further examination as potential therapeutics for a wide variety of diseases, including those related to HPV. |
