This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into RagÀ/À mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAibased cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC 50 for HeLa cells treated with cisplatin was 9.4 M, but after the addition of a lentivirus-delivered shRNA against E6, the IC 50 was reduced almost 4-fold to 2.4 M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.

We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro.

RNA interference (RNAi) is the latest new technology in the field of genetic medicine in which specific genes can be turned off, or silenced, so as to affect a therapeutic outcome. It can be highly specific, works in the nanomolar range and is far more effective than the antisense approaches popular 10-15 years ago. Here we review the field and explore the potential role of RNAi in cancer therapy, highlighting recent progress and examining the hurdles that must be overcome before this promising technology is ready for clinical use.

Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.