Use of chimeric phi29 RNA nanoparticles for delivery of siRNA into colorectal cancer cells

Colorectal cancer is the second leading cause of cancer-related deaths in the United States2. Metastatic or recurrent disease is the most common cause of death in these patients2. Despite the development of various delivery carriers for siRNA, few successful cases have been reported for treating metastatic tumors with systemically delivered siRNA. Previously, we have shown that PI3K-specific siRNA treatment decreased cell viability in vitro and suppressed metastatic tumor growth in vivo3. The goal of this project is to evaluate RNA aptamer conjugated phi29 pRNA three-way junction (3WJ) nanoparticles for the selective delivery of siRNA into colorectal cancer liver metastases via epithelial cell adhesion molecule (EpCAM) in vitro and in vivo. We proposed folate as delivery molecules in round I since we have tested the binding of fluorescent folate-pRNA nanoparticles in vivo using the xenografts4. However, we noted that folate expressivity is variable in colorectal cancer metastases5. Therefore, we turned to use EpCAM (97.7% expression in colon cancer cells) as alternative ligand in the delivery of 3WJ-pRNA/aptamer/siRNA nanoparticles6. Phage phi29 pRNA is used as a vector to carry siRNA sequences7. We tested several metastatic colorectal cancer (CRC) tumor cell lines for EpCAM expression and found that HCT116 cells expressed high levels of EpCAM and exhibited specific binding and internalization of 3WJ-pRNA harboring EpCAM aptamer (Fig. 2). 3WJ-pRNA nanoparticles that contains both EpCAM binding aptamer and siRNAs against firefly luciferase was constructed for specific gene silencing evaluation8-9. Colon cells lines were stably transfected with the GFP and firefly luciferase (Luc) vectors. We evaluated in vitro delivery of 3WJ-pRNA nanoparticle harboring both the EpCAM binding aptamer and the siRNA (firefly luciferase) into EpCAM positive tumor cells with firefly luciferase expression. Next, we will evaluate the in vivo delivery of 3WJ-pRNA nanoparticle. Lastly, we will determine whether PI3K-specific