Systemic targeted EWS-FLI1 siRNA abrogates growth of metastases in a murine Ewing’s tumor model
Authors: Siwen Hu, Jeremy D. Heidel, Derek W. Barlett, Donald B. Kohn, Mark E. Davis, Timothy J. Triche
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

6104 

Despite aggressive therapy, 40% of patients with localized Ewing’s family tumors (EFT) and 80% with metastasis die due to tumor progression. More specific and effective treatment is needed. EWS-FLI1, the chimeric gene product of a chromosome translocation found in more than 90% of EFT, is crucial for maintaining EFT growth. siRNA targeting the breakpoint of EWS-FLI1 (siEFBP) from a pol III promoter reduced the gene transcripts up to 80% and resulted in growth inhibition of the cultured EFT cell line TC135. Since EWS-FLI1 expression is tumor-specific, it serves as an ideal candidate for targeted therapy of EFT. RNAi mediated gene silencing has the potential to be developed into specific, potent and safe cancer therapeutics. However, systemic delivery of siRNA has so far been problematic. Tumor-specific targeting is needed for delivery in high concentration to the tumor sites. Previously described delivery methods, i.e. high-pressure tail injection and viral plasmids, are not feasible for clinical use. Thus, we developed a cyclodextrin (CDP) polymer based delivery system that can be modified with ligands for cell targeting. CDP condenses siRNA into nanoparticles and transferrin (Tf) ligand provides for receptor mediated endocytosis via transferrin receptor typically upregulated in tumor cells. Using this system, we tested that CDP mediated targeted delivery of siEFBP can abrogate EWS-FLI1 expression and inhibit EFT growth in vivo. We verified that CDP-PEG-Tf formulated siEFBP could induce a sequence-specific decrease of EWS-FLI1 protein by more than 50% in cultured TC71 EFT cells. A EFT mouse metastasis model was established with high reproducibility and clinical relevance with TC71 cells constitutively expressing luciferase. An in vivo bioluminescent imaging system (Xenogen) allows the time dependent tumor growth to be monitored in the same mouse. Engraftment sites correspond to the common metastasis sites in EFT patients. Formulated siRNA targeting luciferase (siLUC) resulted in a strong but transient inhibition of luciferase expression in the tumors. Tumor growth was transiently delayed by formulated siEFBP treatment in 3 consecutive days. Most strikingly, long-term formulated siEFBP treatment markedly inhibited EFT growth when administered early, with little or no tumor growth in many animals. Interestingly, the formulated siEFBP does not appear to cross the blood-brain barrier, since a brain metastasis was not affected. Treatments with vehicle alone, siEFBP alone, or formulated unrelated siRNA had no therapeutic effect. This is the first study to show that systemic administration of siRNA can inhibit disseminated tumor growth. We conclude that CDP mediated targeted delivery of siRNA is a powerful and simple method to induce gene silencing in vivo. Future studies using EFT-specific CD99 targeting and combinations with small molecule anti-tumor agents will further enhance the effect of this system.