In-vivo SELEX Against Pancreatic Cancer Xenografts

Abstract

Introduction: Aptamers are single-stranded DNA or RNA oligonucleotides that fold into specific three dimensional shapes and bind with high affinity to protein targets. Aptamers are generated though a process termed Selected Evolution of Ligands by Exponential Enrichment (SELEX). Traditionally, the SELEX process is carried out in vitro by exposing a combinatorial pool of 1013-1015 oligonucleotide sequences to a purified protein target of interest and allowing them to bind. Those sequences that successfully bind are then isolated and amplified by PCR, and the process is repeated over multiple rounds to ultimately yield the sequence with highest affinity for the target. Recently, in vivo SELEX strategies have emerged as novel techniques for specifically identifying cancer targets in the context of a living organism, which may be more relevant for the development of therapeutic agents. Our hypothesis is that in vivo selection against a pancreatic cancer xenograft will identify aptamers that specifically target human pancreatic cancer tissue. Methods: Subcutaneous xenografts were generated in the flanks of nude mice by injection of the BXPC-3 human pancreatic cancer cell line. Two different nuclease resistant RNA libraries (with either 2'fluoro (2'F) or 2'O-methyl (2'OMe) modified pyrimidines) were injected systemically into tumor-bearing mice. After one hour, the tumor tissue was harvested. RNAs that successfully bound to the tissue were isolated and amplified, and the process was repeated. Real-time PCR was performed to determine whether enrichment for tumor-specific aptamers occurred as rounds progressed. After 10 rounds, the pools were clones and sequenced. Results: Real-time PCR appeared to demonstrate enrichment for tumor-binding aptamers as rounds progressed, as demonstrated by an increasing percentage of recovered RNA (Table 1). A few specific sequences were highly represented in the round 10 pools. Clone F-5 represented 39 out of 63 sequences (49%) of the 2'F pool. Clone O-8 represented 14 out of 68 sequences (20.6%) in the 2'OMe pool. Experiments are ongoing to determine which cellular targets these aptamers bind. Conclusions: in vivo SELEX appears to be a feasible technique for selecting aptamers that target tumor tissue and has the potential to identify novel cancer targets of therapeutic interest. Tabled 1 % of input RNA recovered from tumor Round 2'O 2'F 5 0.085% 0.5% 6 0.076% 1.3% 7 0.106% 1.3% 8 0.166% 1.3% 9 0.116% 9.9% Open table in a new tab