Targeting of siRNAs, genes and drugs to cancer cells

DESCRIPTION (provided by applicant): This project aims to identify peptides that can be used to target drugs (small molecules, biologicals, DNA, siRNA) specifically to liver tumor cells. We will use phage display selection techniques to identify such peptides. The incidence of liver cancer, either primary or metastatic, is increasing. Conventional treatments have not been very successful, with reported 5-year survival rates between 20-35%. Thus, there is a great need for improved treatments with better therapeutic indices, by targeting drugs specifically to liver cancer cells. RNA interference is providing a novel means for treating cancer. It is widely recognized that effective delivery of siRNA or siRNA expression vectors is the main hurdle for implementation. Recent work has focused on synthetic delivery vehicles, in which targeting specificity is conveyed by the inclusion of ligands. Our work has shown effective delivery of siRNA to hepatocytes using synthetic delivery vehicles. Many targeting peptides have been identified through the phage display selection technique. However, the liver and liver tumors have so far not been probed by phage display because of technical hurdles. Previous studies have used filamentous phage, which cannot probe extravascular cells. This application proposes to use T7 phage, which are small and can pass through vessel pores after intravenous delivery. The T7 phage display vector is modified to prevent non-specific uptake by liver cells (p17 mutation) or recognition by natural antibodies (low copy number display on truncated p10B). Non-specific phage uptake has previously prevented effective probing of the liver or liver tumors. These studies are designed to provide proof-of-concept for using the modified T7 phage system for selecting liver tumor-targeting peptides in vivo. In Aim 1, a CT26 cell stably expressing green fluorescent protein (GFP) will be generated. A line will be developed with similar growth characteristics as the parent CT26, which forms near-exclusively liver tumors upon injection into the ileocolic vein. Several T7 phage display libraries will be generated, amplified and injected into mice with CT26-GFP liver tumors. Tumor cells will then be isolated from the liver, phage recovered, amplified and re-injected for additional selection rounds. After several selection rounds (4-6), the displayed peptide of individual phage clones will be analyzed. Selections will be performed in mice with small metastasis and larger tumors. The small metastases will require cell sorting to isolate CT26- GFP cells. The larger tumors will be isolated by dissection. In Aim 2, the selection studies will be performed with a human cancer cell line (HT-29, excision of larger tumors only). In Aim 3, the identified peptide sequences will be studied in more detail. Peptides will be aligned for identification of motifs and compared to known sequences. Individual clones will be tested for targeting specificity by biodistribution studies.The incidence of primary and metastatic liver cancer is steadily increasing. The disease is difficult to treat with 5-year survival rates of only 20-35%. Thus, there is a great need for improved therapies. The proposed research will identify ligands that will allow targeting of drugs (small molecules, biologicals, DNA, siRNA) specifically to liver cancer cells, thereby significantly improving the therapeutic index.