71. Intratumoral Delivery of Small Interfering RNA for Inhibition of Tumor Progression in Mice

Abstract

RNA interference (RNAi) is a post-transcriptional gene silencing event in which short double-stranded RNA (siRNA) degrades target mRNA in a sequence-specific manner. Silencing oncogenes or other genes contributing to tumor cell malignancy or progression by siRNA or siRNA-expressing vector offers a therapeutic treatment for cancer patients. For treating cancer patients with RNAi effector, its delivery to tumor cells is one of the key factors, because the gene silencing event is limited in the cells reached by the RNAi effector. To examine whether siRNA or siRNA-expressing vector is effective in reducing target gene expression in tumor cells in vivo, we developed tumor cell lines that stably express reporter genes for a convenient, sensitive and quantitative evaluation of the RNAi effect. Namely, mouse melanoma B16-BL6 cells were stably transfected with firefly (model target gene of RNAi) and sea pansy (indicator of tumor cell number) luciferases to obtain B16-BL6/dual Luc cells. We found that the ratio of the luciferase activities in the tumor cells can be used as an indicator of RNAi. When the siRNA-expressing plasmid DNA (pDNA) was added to the cells, the luciferase activity decreased with time and reached a trough level at 2 days. Then, the activity returned to the initial level at 12 days. siRNA also showed a similar inhibitory profile, but the decrease and recovery in the luciferase activity were faster than those of siRNA-expressing pDNA. Then, the B16-BL6/dual Luc cells were inoculated into the footpad of mice to examine whether the luciferase expression is suppressed by the delivery of RNAi effectors in vivo. A single injection of either siRNA or siRNA-expressing pDNA into the tumor tissue followed by electroporation (1000 V/cm, 5 ms, 4 Hz, 12 pulses) reduced the luciferase activity to about 30% of the control value. Next, we investigated whether silencing the expression of genes related to tumor cell progression by RNAi can inhibit the tumor cell growth. To this end, |[beta]|-catenin and hypoxia-inducible factor 1|[alpha]|(HIF1|[alpha]|) were selected as the target. Transfection of siRNA-expressing pDNA targeting to b-catenin or HIF1a reduced each target gene expression in cultured B16 cells to about 20% (|[beta]|-catenin) and 25% (HIF1|[alpha]|) at mRNA level compared with those in B16 cells transfected with pDNA expressing no effective siRNA. In addition, the treatment resulted in the decrease in the number of B16 cells to about 50% (|[beta]|-catenin) and 70% (HIF1|[alpha]|) of a control value at 3 days. Then we investigated whether the intratumoral delivery of siRNA-expressing pDNA targeting to |[beta]|-catenin or HIF1|[alpha]| can be a therapeutic treatment against the proliferation of the tumor cells in mice. To investigate this possibility, we administered the siRNA-expressing pDNA targeting to |[beta]|-catenin or HIF1|[alpha]| to the tumor-bearing mice by the same procedure as above. As a result, we found that the intratumoral delivery of siRNA-expressing pDNA targeting |[beta]|-catenin or HIF1|[alpha]| inhibited the tumor growth.